Immunogenic compounds for cancer therapy

ABSTRACT

The invention relates to an immunogenic compound comprising an antigenic peptide having amino acid similarity with a tumor antigen, which antigenic peptide is selected in the group consisting of sequences described in the specification.

FIELD OF THE INVENTION

The present invention is in the field of cancer therapy, moreparticularly through immunotherapeutic methods.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death across the world. Accordingto the World Health Organization, in 2012 only, 14 million new cases and8.2 million cancer-related deaths were reported worldwide, and it isexpected that the number of new cancer cases will rise by about 70%within the next two decades. So far, more than 60% of world's total newannual cases occur in Africa, Asia and Central and South America. Theseregions also account for 70% of the world's cancer deaths. Among men,the five most common sites of cancer are lung, prostate, colorectum,stomach and liver; while in women, those are breast, colorectum, lung,cervix, and stomach.

Cancer has long been managed with surgery, radiation therapy, cytotoxicchemotherapy, and endocrine manipulation, which are typically combinedin sequential order so as to best control the disease. However, majorlimitations to the true efficacy of these standard therapies are theirimprecise specificity, which leads to the collateral damage of normaltissues incurred with treatment, a low cure rate, and intrinsic drugresistance.

In the last years, there has been a tremendous increase in thedevelopment of cancer therapies due notably to great advances in theexpression profiling of tumors and normal cells, and recent researchesand first clinical results in immunotherapy, or molecular targetedtherapy, have started to change our perception of this disease.

Promising anticancer immunotherapies have now become a reality andevidences that the host immune system can recognize tumor antigens haveled to the development of anticancer drugs, which are now approved byregulatory agencies as the US Food and Drug Administration (FDA) andEuropean Medicines Agency (EMA). Various therapeutic approaches include,among others, adoptive transfer of ex vivo expanded tumor-infiltratinglymphocytes, cancer cell vaccines, immunostimulatory cytokines andvariants thereof, Pattern recognition receptor (PRR) agonists, andimmunomodulatory monoclonal antibodies targeting tumor antigens orimmune checkpoints (Galuzzi et al., Classification of current anticancerimmunotherapies. Oncotarget. 2014 Dec. 30; 5(24):12472-508).

Unfortunately, a significant percentage of patients can still present anintrinsic resistance to some of these immunotherapies or even acquireresistance during the course of treatment. For example, the three-yearsurvival rate has been reported to be around 20% with the anti-CTLA-4antibody Ipilumumab in unresectable or metastatic melanoma (Snyder etal., Genetic basis for clinical response to CTLA-4 blockade in melanoma.N Engl J Med. 2014 Dec. 4; 371(23):2189-2199; Schadendorf et al., PooledAnalysis of Long-Term Survival Data From Phase II and Phase III Trialsof Ipilimumab in Unresectable or Metastatic Melanoma. J Clin Oncol. 2015Jun. 10; 33(17):1889-94), while the three-year survival rate withanother check point inhibitor, Nivolumab targeting PD1, has beenreported to be of 44% in renal cell carcinoma (RCC) and 18% in NSCLC (McDermott et al., Survival, Durable Response, and Long-Term Safety inPatients With Previously Treated Advanced Renal Cell Carcinoma ReceivingNivolumab. J Clin Oncol. 2015 Jun. 20; 33(18):2013-20; Gettinger et al.,Overall Survival and Long-Term Safety of Nivolumab (Anti-ProgrammedDeath 1 Antibody, BMS-936558, ONO-4538) in Patients With PreviouslyTreated Advanced Non-Small-Cell Lung Cancer. J Clin Oncol. 2015 Jun. 20;33(18):2004-12). Fundamental drug resistance thus represents a fixedbarrier to the efficacy of these immunotherapies. It is thus clear thata different approach to cancer treatment is needed to break thisbarrier.

Absence of response in a large number of subjects treated with theseimmunotherapies might be associated with a deficient anti-tumor immuneresponse (as defect in antigen presentation by APC or antigenrecognition by T cells). In other words, positive response toimmunotherapy correlates with the ability of the immune system todevelop specific lymphocytes subsets able to recognize MHC classI-restricted antigens that are expressed by human cancer cells(Kvistborg et al., Human cancer regression antigens. Curr Opin Immunol.2013 April; 25(2):284-90). This hypothesis is strongly supported by datademonstrating that response to adoptive transfer of tumor-infiltratinglymphocytes, is directly correlated with the numbers of CD8⁺ T-cellstransfused to the patient (Besser et al., Adoptive transfer oftumor-infiltrating lymphocytes in patients with metastatic melanoma:intent-to-treat analysis and efficacy after failure to priorimmunotherapies. Clin Cancer Res. 2013 Sep. 1; 19(17):4792-800). Apotent anti-tumoral response will thus depend on the presentation ofimmunoreactive peptides and the presence of a sufficient number ofreactive cells “trained” to recognize these antigens.

Tumor antigen-based vaccination represent a unique approach to cancertherapy that has gained considerable interest as it can enlist thepatient's own immune system to recognize, attack and destroy tumors, ina specific and durable manner. Tumor cells are indeed known to express alarge number of peptide antigens susceptible to be recognized by theimmune system. Vaccines based on such antigens thus provide greatopportunities not only to improve patient's overall survival but alsofor the monitoring of immune responses and the preparation of GMP-gradeproduct thanks to the low toxicity and low molecular weight of tumorantigens. Examples of tumor antigens include, among others, by-productsof proteins transcribed from normally silent genes or overexpressedgenes and from proteins expressed by oncovirus (Kvistborg et al., Humancancer regression antigens. Curr Opin Immunol. 2013 April;25(2):284-90), and neo-antigens, resulting from point mutations ofcellular proteins. The later are of particular interest as they havebeen shown to be directly associated with increased overall survival inpatient treated with CTLA4 inhibitors (Snyder et al., Genetic basis forclinical response to CTLA-4 blockade in melanoma. N Engl J Med. 2014Dec. 4; 371(23):2189-2199; Brown et al., Neo-antigens predicted by tumorgenome meta-analysis correlate with increased patient survival. GenomeRes. 2014 May; 24(5):743-50).

Nevertheless, the number of human tumor antigens on which cancervaccines can be developed is limited. In particular, antigens derivedfrom mutated or modified self-proteins may induce immune toleranceand/or undesired autoimmunity side effects.

There is thus a need in the art to identify alternative cancertherapeutics, which can overcome the limitations encountered in thisfield, notably resistance to immunotherapies that are currentlyavailable.

The invention has for objective to meet the aforementioned needs.

SUMMARY OF THE INVENTION

The invention relates to an antigenic peptide having amino acidsimilarity with a tumor antigen, which antigenic peptide is selected inthe group consisting of SEQ ID NO:1 to 106. In other words, the presentinvention relates to an antigenic peptide having an amino acid sequenceas set forth in any one of SEQ ID NO:1 to 106. An antigenic peptideaccording to the invention can be in the form of an immunogeniccompound.

Thus, according to certain embodiments, the invention relates to animmunogenic compound comprising an antigenic peptide having amino acidsimilarity with a tumor antigen, which antigenic peptide is selected inthe group consisting of SEQ ID NO:1 to 106, and in particular SEQ IDNO:71. In other words, the present invention relates to an immunogeniccompound comprising an antigenic peptide having an amino acid sequenceas set forth in any one of SEQ ID NO:1 to 106.

More particularly, the invention relates to an immunogenic compound asdefined above, wherein the said antigenic peptide is linked to a carrierprotein.

The present invention relates also to a nanoparticle loaded with atleast antigenic peptide according to the present invention or with atleast one immunogenic compound according to the present invention, and,optionally, with an adjuvant.

The invention also relates to a composition comprising an antigenicpeptide or an immunogenic compound as above defined, the saidcomposition preferably further comprising one or more pharmaceuticallyacceptable excipients.

Thus, according to certain embodiments, the invention relates to animmunogenic composition comprising an antigenic peptide or animmunogenic compound as above defined and one or more pharmaceuticallyacceptable excipients,

Preferably, the said immunogenic composition may further comprise one ormore immunostimulatory agents.

The said one or more immunostimulatory agents may be selected in a groupcomprising (or consisting of) immuno-adjuvants and antigen-presentingcells.

The said antigen-presenting cells may consist of dendritic cells.

According to other embodiments, the invention relates to an antigenicpeptide as above defined or an immunogenic compound as above defined,for use in the prevention or in the treatment of a cancer.

According to further embodiments, the invention relates to animmunogenic composition for use in the prevention or in the treatment ofa cancer.

This invention also pertains to the use of an antigenic peptide as abovedefined or of an immunogenic compound as above defined, for preparing amedicament for treating or preventing a cancer.

This invention also concerns a method for preventing or treating acancer in an individual in need thereof, wherein the said methodcomprises a step of administering to the said individual an antigenicpeptide as above defined or an immunogenic compound as above defined oran immunogenic composition as above defined or a nanoparticle accordingto the present invention or a nucleic acid according to the presentinvention or a combination according to the present invention.

According to yet further embodiments, the invention relates to a nucleicacid coding for an antigenic peptide or an immunogenic compound as abovedefined.

Furthermore, the present invention also relates to a combination of twodistinct immunogenic compounds according to the present invention foruse in the prevention and/or treatment of a cancer. Furthermore, thepresent invention also relates to a combination of two distinctantigenic peptides according to the present invention for use in theprevention and/or treatment of a cancer. Furthermore, the presentinvention also relates to a combination of two distinct nanoparticlesaccording to the present invention for use in the prevention and/ortreatment of a cancer. Furthermore, the present invention also relatesto a combination of two distinct nucleic acids according to the presentinvention for use in the prevention and/or treatment of a cancer.

In certain embodiments, the two distinct components of the combinationfor use according to the present invention are comprised in the same ordistinct compositions.

In certain embodiments, the two distinct components of the combinationfor use according to the present invention are administered via the sameor distinct routes of administration.

In certain embodiments, the two distinct components of the combinationfor use according to the present invention are administered at about thesame time or consecutively.

Furthermore, the present invention also relates to a kit comprising

-   -   an immunogenic compound according to the present invention,    -   an antigenic peptide according to the present invention,    -   a nanoparticle according to the present invention,    -   a nucleic acid according to the present invention, or    -   an immunogenic composition according to the present invention.

DESCRIPTION OF THE FIGURES

FIG. 1: General protocol for the validation of the Proof-of-concept of atumor antigen-based immunotherapy targeting IL13RA2.

FIG. 2: Schematic view of the Immunization scheme. d: day.

FIG. 3: ELISPOT-IFNγ results for group 1 (IL13RA2-B) and group 2(IL13RA2-A). The peptide used for vaccination (in between brackets undereach group) and the stimulus used in the ELISPOT culture (X-axis) areindicated on the graphs. (A) Number of specific ELISPOT-IFNγ spots(medium condition subtracted). Each dot represents the average value forone individual/mouse from the corresponding condition quadruplicate. (B)For each individual, the level of specific ELISPOT-IFNγ response iscompared to the ConA stimulation (value: 100%). Statistical analysis:paired t-test for intra-group comparison and unpaired t-test forinter-group comparison; * p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have identified a set of antigenic peptides that can beused to induce a specific immune response against tumor cells.

Those antigenic peptides all share the property of having amino acidsimilarity with tumor antigens encoded by the set of genes disclosed inTable 1A and Table 1B.

For instance, the Interleukin-13 receptor subunit alpha-2 (IL-13Rα2 orIL13RA2) is a membrane bound protein that in humans is encoded by theIL13RA2 gene. In a non-exhaustive manner, IL13RA2 has been reported as apotential immunotherapy target (see Beard et al.; Clin Cancer Res;72(11); 2012). The high expression of IL13RA2 has further beenassociated with invasion, liver metastasis and poor prognosis incolorectal cancer (Barderas et al.; Cancer Res; 72(11); 2012).

Accordingly, the invention relates to antigenic peptides having aminoacid similarity with a tumor antigen, which antigenic peptide isselected in the group consisting of SEQ ID NO:1 to 106.

The expression “having amino acid similarity with a tumor antigen” asused herein, refer in particular to sequence variants of fragments of a(reference) tumor antigen, such as IL13RA2.

A sequence variant shares, in particular over the whole length of thesequence, at least 50% sequence identity with a reference sequence,namely, a fragment of a (reference) tumor antigen. Preferably, thesequence variant shares at least 60%, preferably at least 70%,preferably at least 75%, more preferably at least 80%, even morepreferably at least 85%, still more preferably at least 90%,particularly preferably at least 95%, and most preferably at least 99%sequence identity with a reference sequence, namely, a fragment of a(reference) tumor antigen. Sequence identity may be calculated asdescribed below. Preferably, a sequence variant preserves the specificfunction of the reference sequence, for example its function as epitope.In particular, an amino acid sequence variant has an altered sequence inwhich one or more of the amino acids in the reference sequence isdeleted or substituted, or one or more amino acids are inserted into thesequence of the reference amino acid sequence. For example, variantsequences, which are at least 90% identical, have no more than 10alterations, i.e. any combination of deletions, insertions orsubstitutions, per 100 amino acids of the reference sequence.

Methods for comparing the identity (similarity) of two or more sequencesare well known in the art. The percentage to which two sequences areidentical can, e.g., be determined using a mathematical algorithm. Apreferred, but not limiting, example of a mathematical algorithm, whichcan be used, is the algorithm of Karlin et al. (1993), PNAS USA,90:5873-5877. Such an algorithm is integrated in the BLAST family ofprograms, e.g. BLAST or NBLAST program (see also Altschul et al., 1990,J. Mol. Biol. 215, 403-410 or Altschul et al. (1997), Nucleic Acids Res,25:3389-3402), accessible through the home page of the NCBI at worldwide web site ncbi.nlm.nih.gov) and FASTA (Pearson (1990), MethodsEnzymol. 183, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci.U.S.A 85, 2444-2448). Sequences that are identical to other sequences toa certain extent can be identified by these programmes. Furthermore,programs available in the Wisconsin Sequence Analysis Package, version9.1 (Devereux et al., 1984, Nucleic Acids Res., 387-395), for examplethe programs BESTFIT and GAP, may also be used to determine the %identity between two polynucleotides and the % identity between two(poly)peptide sequences. BESTFIT uses the “local homology” algorithm ofSmith and Waterman (1981), J. Mol. Biol. 147, 195-197 and finds the bestsingle region of similarity between two sequences.

The “fragment” of the (reference) tumor antigen, which typically servesas reference sequence, comprises at least seven, preferably at leasteight and most preferably (at least) nine amino acids or ten aminoacids.

Advantageously, those antigenic peptides may be in the form ofimmunogenic compounds, in particular for use in the prevention or in thetreatment of a cancer.

Thus, the invention also relates to an immunogenic compound comprisingan antigenic peptide having amino acid similarity with a tumor antigen,which antigenic peptide is selected in the group consisting of SEQ IDNO:1 to 106. In other words, the present invention provides an antigenicpeptide having an amino acid sequence as set forth in any one of SEQ IDNO:1 to 106. Preferably, the present invention provides (an immunogeniccompound comprising) an antigenic peptide comprising or consisting of anamino acid sequence as set forth in any one of SEQ ID NOs: 17, 31, 32,51, 52, 55, 56, 59, 68, 89, 94, 100, 101 or 102. It is also preferredthat the present invention provides (an immunogenic compound comprising)an antigenic peptide comprising or consisting of an amino acid sequenceas set forth in any one of SEQ ID NOs: 26, 28, 47, 51, 52, 55, 56, 77,93, 101 or 102. More preferably, the present invention provides (animmunogenic compound comprising) an antigenic peptide comprising orconsisting of an amino acid sequence as set forth in any one of SEQ IDNOs: 51, 52, 55, 56, 101 or 102. Even more preferably, the presentinvention provides (an immunogenic compound comprising) an antigenicpeptide comprising or consisting of an amino acid sequence as set forthin any one of SEQ ID NOs: 51, 52, 55 or 56. It is also even morepreferred that the present invention provides (an immunogenic compoundcomprising) an antigenic peptide comprising or consisting of an aminoacid sequence as set forth in any one of SEQ ID Nos: 101 or 102.

As shown in the examples herein, the said specific antigenic peptidesaccording to the present invention allow the raise of a strong immuneresponse against themselves, and most importantly, allow the raise of astrong immune response against peptides having amino acid similaritytherewith which are comprised in the IL13RA2 tumor antigen, although thesaid peptides comprised in the IL13RA2 tumor antigen are themselvestolerogenic.

Without wishing to be bound by any particular theory, the inventorsbelieve that the high expression of gamma interferon which has beenmeasured after an in vivo administration of an immunogenic compositioncomprising an antigenic peptide described herein illustrates theactivation of antigenic peptide-specific T-cells, and especially theactivation of antigenic peptide-specific CTLs, which cells are known inthe art to be relevant immune effectors of an anti-cancer immuneresponse.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, nomenclatures used herein, andtechniques of cell and tissue culture are those well-known and commonlyused in the art.

Such techniques are fully explained in the literature, such as Owen etal. (Kuby Immunology, 7^(th), edition, 2013—W. H. Freeman) and Sambrooket al. (Molecular cloning: A laboratory manual 4th edition, Cold SpringHarbor Laboratory Press—Cold Spring Harbor, N.Y., USA, 2012).

Nevertheless, with respect to the use of different terms throughout thecurrent specification, the following definitions more particularlyapply.

The terms “peptide”, “polypeptide” and “protein” refer herein to asequential chain of amino acids of any length linked together viapeptide bonds (—NHCO—), and which can play a structural and/orfunctional role in a cell in vitro and/or in vivo. It encompasses aminoacids chains in size ranging from 2 to at least about 1000 amino acidresidues. The term “peptide” preferably encompasses herein amino acidchains in size of less than about 30 amino acids, while the terms“polypeptide” and “protein” preferably encompass amino acid chains insize of at least 30 amino acids. The terms “polypeptide” and “protein”are used herein interchangeably. As well-known in the art, peptides,polypeptides and proteins can be encoded by nucleic acids.

The term “antigenic peptide” refers to a peptide, which preferably hasamino acid similarity with a tumor protein, and which is prone to induceor maintain an immunological response against said peptide in a subjectto whom it is administered.

The term “immunogenic compound” refers to a compound comprising anantigenic peptide as defined above, which is also able to induce ormaintain an immunological response against said peptide from the subjectfor whom it is administered.

In some embodiments, immunogenic compounds comprise at least oneantigenic peptide, or alternatively at least one compound comprisingsuch an antigenic peptide, linked to a protein, which encompasses acarrier protein.

A carrier protein is usually a protein, which is able to transport acargo, such as the antigenic peptide according to the present invention.For example, the carrier protein may transport its cargo across amembrane. In the context of the present invention, a carrier protein inparticular (also) encompasses a peptide or a polypeptide that is able toelicit an immune response against the antigenic peptide that is linkedthereto. Carrier proteins are known in the art.

In some embodiments, an antigenic peptide as described herein, or apolypeptide comprising the said antigenic peptide, may be linked, forexample by covalent or non-covalent bond, to a protein havingimmuno-adjuvant properties, such as the HHD-DR3 peptide of sequenceMAKTIAYDEEARRGLERGLN (SEQ ID NO:144).

Alternatively such carrier peptide or polypeptide may be co-administeredin the form of immune adjuvant.

Preferably, the antigenic peptide as described herein, or a polypeptidecomprising the antigenic peptide, may be co-administrated or linked, forexample by covalent or non-covalent bond, to a protein/peptide havingimmuno-adjuvant properties, such as providing stimulation of CD4+ Th1cells. While the antigenic peptide as described herein preferably bindsto MHC class I, CD4+ helper epitopes may be additionally used to providean efficient immune response. Th1 helper cells are able to sustainefficient dendritic cell (DC) activation and specific CTL activation bysecreting interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α)and interleukine-2 (IL-2) and enhancing expression of costimulatorysignal on DCs and T cells (Galaine et al., Interest of Tumor-SpecificCD4 T Helper 1 Cells for Therapeutic Anticancer Vaccine. Vaccines(Basel). 2015 Jun. 30; 3(3):490-502).

For example, the adjuvant peptide/protein may preferably be a non-tumorantigen that recalls immune memory or provides a non-specific help orcould be a specific tumor-derived helper peptide. Several helperpeptides have been described in the literature for providing anonspecific T cell help, such as tetanus helper peptide, keyhole limpethemocyanin peptide or PADRE peptide (Adotevi et al., Targeting antitumorCD4 helper T cells with universal tumor-reactive helper peptides derivedfrom telomerase for cancer vaccine. Hum Vaccin Immunother. 2013 May;9(5):1073-7, Slingluff C L, The present and future of peptide vaccinesfor cancer: single or multiple, long or short, alone or in combination?Cancer J. 2011 September-October; 17(5):343-50). Accordingly, tetanushelper peptide, keyhole limpet hemocyanin peptide and PADRE peptide arepreferred examples of such adjuvant peptide/proteins. Moreover, specifictumor derived helper peptides are preferred. Specific tumor derivedhelper peptides are typically presented by MHC class II, in particularby HLA-DR, HLA-DP or HLA-DQ. Specific tumor derived helper peptides maybe fragments of sequences of shared overexpressed tumor antigens, suchas HER2, NY-ESO-1, hTERT or IL13RA2. Such fragments have preferably alength of at least 10 amino acids, more preferably of at least 11 aminoacids, even more preferably of at least 12 amino acids and mostpreferably of at least 13 amino acids. In particular, fragments ofshared overexpressed tumor antigens, such as HER2, NY-ESO-1, hTERT orIL13RA2, having a length of 13 to 24 amino acids are preferred.Preferred fragments bind to MHC class II and may, thus, be identifiedusing, for example, the MHC class II binding prediction tools of IEDB(Immune epitope database and analysis resource; Supported by a contractfrom the National Institute of Allergy and Infectious Diseases, acomponent of the National Institutes of Health in the Department ofHealth and Human Services; www.iedb.org/; http://tools.iedb.org/mhcii/.

A composition as defined herein which comprises an immunogenic compoundas defined above, and which further comprises one or moreimmuno-adjuvant substances, may also be termed an “immunogeniccomposition” or in some embodiments a “vaccine composition” in thepresent specification.

As used herein, the term “immunogenic composition” refers to acomposition that is able to induce or maintain an immune response, inparticular which induces an immune response, when it is administered toa mammal, and especially when it is administered to a human individual.

The terms “nucleic acid”, “nucleic acid molecule”, “nucleic acidsequence”, “polynucleotide”, “nucleotide sequence”, which are usedherein interchangeable, refer to a precise succession of naturalnucleotides (e.g., A, T, G, C and U), or synthetic nucleotides,corresponding to a single-stranded or double-stranded DNA or RNA, suchas cDNA, genomic DNA, ribosomal DNA, and the transcription product ofsaid DNA, such as RNA, rRNA, mRNA; antisense DNA, antisense RNA;complementary RNA and/or DNA sequences; RNA and/or DNA sequences with orwithout expression elements, regulatory elements, and/or promoters; avector; and combinations thereof. It is within the skill of the personin the art to determine nucleotide sequences that can encode a specificamino acid sequence.

The (poly)peptides and/or nucleic acids according to the invention maybe prepared by any known method in the art including, but not limitedto, any synthetic method, any recombinant method, any ex vivo generationmethod and the like, and any combination thereof. Such techniques arefully explained in the literature as mentioned above.

In the context of the present invention, the antigenic peptidesaccording to the invention comprise antigens having similarity with atumor antigen. As used herein, the term “tumor antigen” comprisestumor-specific antigens and tumor-associated antigens. In general, theterm “tumor antigen” or “tumor protein” designates herein an antigenicsubstance produced in tumor cells, and sometimes also in normal cells,and which can trigger an immune response upon administration in asubject. In humans, those have been classified according to theirexpression pattern, function or genetic origin, and include withoutlimitation, overexpressed self-antigens (such as HER2/neu and itsvariant dHER2, p53, Wilm's Tumor 1, Ephrin receptor, Proteinase-3,Mucin-1, Mesothelin, EGFR, CD20); cancer-testis (CT) antigens (such asMAGE-1, BAGE, GAGE, NY-ESO-1); mutational antigens, also known asneo-antigens (such as mutants from MUM-1, bcr-abl, ras, b-raf, p53,CDK-4, CDC27, beta-catenin, alpha-actenin-4); tissue-specificdifferentiation antigens (such as the melanoma antigens Melan A/MART-1,tyrosinase, TRP1/pg75, TRP2, gp100 and gangliosides GM3, GM2, GD2 andGD3; the prostate cancer antigens PSMA, PSA and PAP); viral antigenswhich are expressed by oncoviruses (such as HPV, EBV); oncofetalantigens (such as alphafetoprotein AFP and carcinoembryonic antigenCEA); and universal antigens (telomerase, hTERT, survivin, mdm-2,CYP-1B1) (Srinivasan and Wolchok, Tumor antigens for cancerimmunotherapy: therapeutic potential of xenogeneic DNA vaccines. JTransl Med. 2004 Apr. 16; 2(1):12).

According to the different aspects and embodiments of the inventiondescribed herein, a “subject” or “host” preferably refers to a mammal,and most preferably to a human being. Said subject may have, beensuspected of having, or be at risk of developing cancer, for examplemelanoma, colorectal cancer or clear cell renal cell carcinoma.

By “pharmaceutically acceptable excipient”, it is meant herein acompound of pharmaceutical grade which improves the delivery, stabilityor bioavailability of an active agent, and can be metabolized by, and isnon-toxic to, a subject to whom it is administered. Preferred excipientsaccording to the invention include any of the excipients commonly usedin pharmaceutical products, such as, for example, water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable excipients may further comprise minoramounts of auxiliary substances such as wetting or emulsifying agents,or preservatives.

By “vaccine”, it is meant herein a composition capable of stimulatingthe immune system of a living organism so that protection against aharmful antigen is provided, either through prophylaxis or throughtherapy.

The term “cancer”, as used herein, refers to a malignant neoplasm. Inparticular, the term “cancer” refers herein to any member of a class ofdiseases or disorders that are characterized by uncontrolled division ofcells and the ability of these cells to invade other tissues, either bydirect growth into adjacent tissue through invasion or by implantationinto distant sites by metastasis. Metastasis is defined as the stage inwhich cancer cells are transported through the bloodstream or lymphaticsystem. It encompasses, among others, esophageal cancer, gastric cancer,duodenal cancer, small intestinal cancer, appendiceal cancer, largebowel cancer, colon cancer, rectum cancer, colorectal cancer, analcancer, pancreatic cancer, liver cancer, gallbladder cancer, spleencancer, renal cancer, bladder cancer, prostatic cancer, testicularcancer, uterine cancer, endometrial cancer, ovarian cancer, vaginalcancer, vulvar cancer, breast cancer, pulmonary cancer, thyroid cancer,thymus cancer, brain cancer, nervous system cancer, oral cavity cancer,skin cancer, blood cancer, lymphomas, eye cancer, bone cancer, bonemarrow cancer, muscle cancer, etc. . . . . In the context of the presentinvention, melanoma, head and neck, breast, colorectal cancer or clearcell renal cell carcinoma are preferred.

As used herein, the term “preventing”, “prevention”, “prophylaxis” or“prevent” generally means to avoid or minimize the onset or developmentof a disease or condition before its onset, while the term “treating,“treatment” or “treat” encompasses reducing, ameliorating or curing adisease or condition (or symptoms of a disease or condition) after itsonset. In the context of the invention, the prevention and/or treatmentof cancer can lead, for example, to the non-proliferation, weak, reducedor delayed proliferation of tumor cells within the subject, or to thecomplete or almost complete elimination of tumor cells within thesubject. The term “preventing” encompasses “reducing the likelihood ofoccurrence of” or «reducing the likelihood of reoccurrence».

An “effective amount” or “effective dose” as used herein is an amountwhich provides the desired effect. For therapeutic purposes, aneffective amount is an amount sufficient to provide a beneficial ordesired clinical result. The preferred effective amount for a givenapplication can be easily determined by the skilled person taking intoconsideration, for example, the size, age, weight of the subject, thetype of cancer to be prevented or treated, and the amount of time sincethe cancer began. In the context of the present invention, in terms ofprevention or treatment, an effective amount of the composition is anamount that is sufficient to induce a humoral and/or cell-mediatedimmune response directed against cancer.

As used herein, the term “comprising” encompasses “consisting of”.

Additional definitions are provided throughout the specification.

The present invention may be understood more readily by reference to thefollowing detailed description, including preferred embodiments of theinvention, and examples included herein.

Thus, the invention relates to an immunogenic compound comprising anantigenic peptide having amino acid similarity with a tumor antigen,which antigenic peptide is selected in the group consisting of SEQ IDNO:1 to 106. In other words, the present invention provides (animmunogenic compound comprising) an antigenic peptide having an aminoacid sequence as set forth in any one of SEQ ID NO:1 to 106. Preferably,the present invention provides (an immunogenic compound comprising) anantigenic peptide comprising or consisting of an amino acid sequence asset forth in any one of SEQ ID NOs: 17, 31, 32, 51, 52, 55, 56, 59, 68,89, 94, 100, 101 or 102. It is also preferred that the present inventionprovides (an immunogenic compound comprising) an antigenic peptidecomprising or consisting of an amino acid sequence as set forth in anyone of SEQ ID NOs: 26, 28, 47, 51, 52, 55, 56, 77, 93, 101 or 102. Morepreferably, the present invention provides (an immunogenic compoundcomprising) an antigenic peptide comprising or consisting of an aminoacid sequence as set forth in any one of SEQ ID NOs: 51, 52, 55, 56, 101or 102. Even more preferably, the present invention provides (animmunogenic compound comprising) an antigenic peptide comprising orconsisting of an amino acid sequence as set forth in any one of SEQ IDNOs: 51, 52, 55 or 56. It is also even more preferred that the presentinvention provides (an immunogenic compound comprising) an antigenicpeptide comprising or consisting of an amino acid sequence as set forthin any one of SEQ ID NOs: 101 or 102.

According to an exemplary embodiment, the antigenic peptide as abovedefined is a peptide of sequence SEQ ID NO:71.

According to one embodiment, the antigenic peptide as above defined, ora polypeptide comprising the said antigenic peptide, is linked to acarrier protein, for example by a covalent or non-covalent bond.

According some embodiments, the invention relates to an immunogeniccompound as above defined, comprising an antigenic peptide of formula(I):PepNt-CORE-PepCt  (I), wherein:

-   -   “PepNt” consists of a polypeptide having an amino acid length        varying from 0 to 30 amino acid residues and located at the        N-terminal end of the polypeptide of formula (I);    -   CORE consists of a polypeptide comprising, or alternatively        consisting of, an amino acid sequence selected from the group        consisting of SEQ ID NO:1 to 106 (which includes SEQ ID NO:71),        in particular an amino acid sequence as set forth in any one of        SEQ ID NOs: 26, 28, 47, 51, 52, 55, 56, 77, 93, 101 or 102 or an        amino acid sequence as set forth in any one of 17, 31, 32, 51,        52, 55, 56, 59, 68, 89, 94, 100, 101 or 102, such as an amino        acid sequence as set forth in any one of SEQ ID NOs: 51, 52, 55,        56, 101 or 102; and    -   “PepCt” consists of a polypeptide having an amino acid length        varying from 0 to 30 amino acid residues and located at the        C-terminal end of the polypeptide of formula (I).

Preferably, the antigenic peptide of formula (I) is a fusion peptide orfusion protein, in particular a recombinant fusion peptide or protein.The term “recombinant” means that it does not occur in nature.

The invention further relates to a nanoparticle loaded with

-   -   at least one of the immunogenic compounds according to the        present invention, or    -   at least one of the antigenic peptides according to the present        invention;

and, optionally, with an adjuvant

The invention further relates to an immunogenic composition comprising

-   -   an immunogenic compound according to the present invention,    -   an antigenic peptide according to the present invention,    -   a nanoparticle according to the present invention, or    -   a nucleic acid according to the present invention,

and one or more pharmaceutically acceptable excipients.

The immunogenic composition may further comprise one or moreimmunostimulatory agents.

In particular, the said immunostimulatory agent is selected in a groupconsisting of immuno-adjuvants and antigen-presenting cells.

More particularly, the antigen-presenting cells may consist of dendriticcells.

In particular, the immunogenic composition may comprise

(i) two distinct immunogenic compounds according to the presentinvention;

(ii) two distinct antigenic peptides according to the present invention;

(iii) two distinct nanoparticle according to the present invention; or

(iv) two distinct nucleic acid according to the present invention.

In this context, the two distinct components refer in particular todistinct antigenic peptides according to the present invention (whichare comprised by the immunogenic compounds, the nanoparticles and/or thenucleic acids). Such two distinct components, in particular the twodistinct antigenic peptides according to the invention (comprised in thetwo distinct components), relate preferably to the same type of cancer,for example to the same or distinct antigens associated with this cancerand/or to the same or distinct (reference) epitopes within an antigenassociated with this cancer. More preferably, the two distinctcomponents, in particular the two distinct antigenic peptides accordingto the invention (comprised in the two distinct components), relate tothe same tumor (associated or specific) antigen. The two distinctcomponents, in particular the two distinct antigenic peptides accordingto the invention (comprised in the two distinct components), may alsorelate to the same or distinct (reference) tumor (associated orspecific) antigen(s).

The invention further relates to any one of

-   -   the immunogenic compound according to the present invention,    -   the antigenic peptide according to the present invention,    -   the (host) cell according to the present invention,    -   the nanoparticle according to the present invention,    -   the nucleic acid according to the present invention, or    -   the immunogenic composition according to the present invention,

for use in the prevention or in the treatment of a cancer.

Among the different types of cancer, those which are more particularlyconsidered for treatment and/or prevention, are detailed in Table 1Bhere below, in particular in view of the targeted tumor antigen.

TABLE 1B list of therapeutic indications associated with each gene GeneName (Table 1A) Cancers in which of the gene is involved PLIN2 Diseasesassociated with PLIN2 include lipid-rich carcinoma and acrodermatitisenteropathica and colorectal cancer ALDH1A1 Diseases associated withALDH1A1 include lung cancer (including lung adenoma) and breast cancerAFP Diseases associated with AFP include liver cancer, hepatocellularcancer PTPRC Breast cancer CEACAM5 Diseases associated with CEACAM5include gut carcinoma, colorectal cancer, urachal cancer,gastrointestinal cancer and pancreatic cancer ENAH Breast cancer EZH2Diseases associated with EZH2 include many forms of cancers, includinglung cancer and lymphoblastoma PMEL Melanoma ERBB2 Diseases associatedwith ERBB2 include numerous cancers, including breast cancer, glioma andovarian cancer IL13RA2 Diseases associated with IL13RA2 includecolorectal cancer, ovarian cancer, testis cancer, renal cell carcinoma,prostate cancer, glioma, head and neck cancer, astrocytoma, melanoma andbreast cancer metastasis MAGEA1 Diseases associated with MAGEA1 includemelanoma and hemangioma of liver, non-small cell lung cancer, gastriccancer and melanoma MAGEA3 Diseases associated with MAGEA3 include manycancers, including renal cell carcinoma, bladder carcinoma, melanoma,non-small cell lung cancer, hematologic malignancies, among othersMAGEA4 Diseases associated with MAGEA4 include melanoma and testicularleukemia, thyroid cancer, breast cancer including estrogen receptornegative breast cancer and non-small cell lung cancer MAGEC1 Diseasesassociated with MAGEC1 include breast cancer, ovarian carcinoma andprostate cancer MAGEC2 Diseases associated with MAGEC2 includehepatocellular carcinoma, melanoma gastrointestinal stromal tumors,breast cancer metastasis and prostate cancer SCGB2A2 Diseases associatedwith SCGB2A2 include breast cancer MLANA Diseases associated with MLANAinclude melanoma MDK Diseases associated with MDK include multiplecancer types, including breast cancer, thyroid cancer, pancreaticcancer, neuroblastoma, glioblastoma, Wilms' tumors, thyroid papillarycarcinomas, colorectal, liver, ovary, bladder, breast, lung, esophageal,stomach, and prostate cancers MMP2 Diseases associated with MMP2 includemany forms of cancer, including bladder cancer, colorectal, melanoma,breast cancer, lung cancer, ovarian cancer, and prostate cancer CTAG1BDiseases associated with CTAG1B include many cancers, including breastcancer, thyroid cancer, ovarian cancer, melanomas, sarcomas, lungcancer, head and neck cancer, prostate cancer, and bladder cancer ACPPDiseases associated with ACPP include prostate cancer, ovarian cancerand prostatic adenoma STEAP1 Diseases associated with STEAP1 includeprostate cancer TAG1 Diseases associated with TAG1 include brain cancer,breast cancer, colon cancer, lung cancer, ovary cancer, pharynx cancer,tongue cancer, bladder cancer (including urothelial carcinoma of thebladder) TYR Diseases associated with TYR include skin cancer andmelanoma

Thus, according to one embodiment, the invention relates to any one ofthe antigenic peptides and immunogenic compounds described herein, aswell as to any one of the immunogenic compositions described herein, foruse in the prevention or in the treatment of a cancer selected fromTable 1B.

The invention further relates to a nucleic acid coding for an antigenicpeptide having amino acid similarity with a tumor antigen, wherein thepeptide is selected in the group consisting of

-   -   antigenic peptides selected in the group consisting of SEQ ID        NO:1 to 106; and/or    -   antigenic peptides of formula (I), or (Ia), or (Ib), as        described herein.

In particular, the nucleic acid as defined above may code for anantigenic peptide selected in the group consisting of peptides havingamino acid similarity with IL13RA2, which includes SEQ ID NO:71.

The invention also concerns a method for preventing or treating a canceror initiating, enhancing or prolonging an anti-tumor-response in asubject in need thereof comprising administering to the subject anantigenic peptide according to the present invention or an immunogeniccompound according to the present invention or an immunogeniccomposition according to the present invention or a nanoparticleaccording to the present invention or a nucleic acid according to thepresent invention or a combination according to the present invention.

Furthermore, the invention relates to a nucleic acid coding for anantigenic peptide or an immunogenic compound as above defined.

Furthermore, the present invention also relates to a combination of twodistinct immunogenic compounds according to the present invention foruse in the prevention and/or treatment of a cancer.

Furthermore, the present invention also relates to a combination of twodistinct antigenic peptides according to the present invention for usein the prevention and/or treatment of a cancer.

Furthermore, the present invention also relates to a combination of twodistinct nanoparticles according to the present invention for use in theprevention and/or treatment of a cancer.

Furthermore, the present invention also relates to a combination of twodistinct nucleic acids according to the present invention for use in theprevention and/or treatment of a cancer.

In certain embodiments, the two distinct components of the combinationfor use according to the present invention are comprised in the same ordistinct compositions.

In certain embodiments, the two distinct components of the combinationfor use according to the present invention are administered via the sameor distinct routes of administration.

In certain embodiments, the two distinct components of the combinationfor use according to the present invention are administered at about thesame time (simultaneously) or consecutively.

Furthermore, the present invention also relates to a kit comprising

-   -   an immunogenic compound according to the present invention,    -   an antigenic peptide according to the present invention,    -   a (host) cell according to the present invention,    -   a nanoparticle according to the present invention,    -   a nucleic acid according to the present invention, or    -   an immunogenic composition according to the present invention.

Antigenic Peptides, Immunogenic Compounds, Nucleic Acids, Nanoparticlesand Cells

Unless reference to the contrary, all the passages referring to«antigenic peptides» may also be applied to «immunogenic compounds».

Antigenic peptides according to the invention are listed in Table 1Abelow, which also provides information regarding the corresponding“reference” human tumor antigens (epitopes) with the name of the geneencoding them, and in a non-limitative manner their reportedlocalization in tumors. N.A.=Not Available. The sequence IDs SEQ ID NO:1to 106 refer to the antigenic peptide.

TABLE 1A  Antigenic peptides according to the invention SEQ Gene IDcoding for Antigenic NO. antigen Peptide Reference Tumor localization 1PLIN2 SLAGTITGV SVASTITGV adipocytes, macrophages 2 ALDH1A1 LLMKLADLVLLYKLADLI mucosa, keratinocytes 3 ALDH1A1 LLYKIADLV LLYKLADLImucosa, keratinocytes 4 AFP SLALSVILRV QLAVSVILRV liver 5 AFP SLAVSVILRAQLAVSVILRV liver 6 PTPRC KLLDAVISL KFLDALISLproliferating cells, testis, multiple tissues (low level) 7 PTPRCKLLDALLSL KFLDALISL proliferating cells, testis,multiple tissues (low level) 8 PTPRC KMLDALIDL KFLDALISLproliferating cells, testis, multiple tissues (low level) 9 PTPRCKILDSLISL KFLDALISL proliferating cells, testis,multiple tissues (low level) 10 PTPRC KFLDALIGV KFLDALISLproliferating cells, testis, multiple tissues (low level) 11 PTPRCKFLDSLISV KFLDALISL proliferating cells, testis,multiple tissues (low level) 12 CEACAM5 GVLAGVALV GVLVGVALIgut carcinoma 13 CEACAM5 GMLVGVALI GVLVGVALI gut carcinoma 14 CEACAM5GLLMGVALI GVLVGVALI gut carcinoma 15 CEACAM5 GVLVGLALV GVLVGVALIgut carcinoma 16 CEACAM5 GVLAGIALI GVLVGVALI gut carcinoma 17 CEACAM5GILVGVALV GVLVGVALI gut carcinoma 18 CEACAM5 GLLIGVALI GVLVGVALIgut carcinoma 19 CEACAM5 GVLLGVALV GVLVGVALI gut carcinoma 20 CEACAM5GVLTGIALI GVLVGVALI gut carcinoma 21 CEACAM5 GILVGLALI GVLVGVALIgut carcinoma 22 CEACAM5 GVIVGVALV GVLVGVALI gut carcinoma 23 CEACAM5GVFVGLALI GVLVGVALI gut carcinoma 24 CEACAM5 GVLIGVALV GVLVGVALIgut carcinoma 25 CEACAM5 YLFGHSWYK HLFGYSWYK gut carcinoma 26 ENAHTMNGKSSPV TMNGSKSPV breast, prostate stroma andepithelium of colon-rectum, pancreas, endometrium 27 EZH2 FMAEDETLLFMVEDETVL ubiquitous (low level) 28 PMEL ITSDVPFSV ITDQVPFSV melanoma 29ERBB2 IMSAVIGIL IISAVVGIL ubiquitous (low level) 30 ERBB2 ILSAVIGILIISAVVGIL ubiquitous (low level) 31 ERBB2 ILSAVVGVL IISAVVGILubiquitous (low level) 32 ERBB2 IMSAVVGIL IISAVVGILubiquitous (low level) 33 ERBB2 FISAVVGVL IISAVVGILubiquitous (low level) 34 ERBB2 ILSAVVGIL IISAVVGILubiquitous (low level) 35 ERBB2 IISAVIGIV IISAVVGILubiquitous (low level) 36 ERBB2 IISAIVGLL IISAVVGILubiquitous (low level) 37 ERBB2 IISAIVGIV IISAVVGILubiquitous (low level) 38 ERBB2 IISAVVGVV IISAVVGILubiquitous (low level) 39 ERBB2 IISAVVGIV IISAVVGILubiquitous (low level) 40 ERBB2 LISAVVGLL IISAVVGILubiquitous (low level) 41 ERBB2 ILYGGAYSL ILHNGAYSLubiquitous (low level) 42 ERBB2 KLYGSLAFL KIFGSLAFLubiquitous (low level) 43 ERBB2 KIFGTLAFM KIFGSLAFLubiquitous (low level) 44 ERBB2 PLADIISAV PLTSIISAVubiquitous (low level) 45 ERBB2 PLASIFSAV PLTSIISAVubiquitous (low level) 46 ERBB2 PLSSILSAV PLTSIISAVubiquitous (low level) 47 ERBB2 RLLEETDLV RLLQETELVubiquitous (low level) 48 ERBB2 TLNDITGYL TLEEITGYLubiquitous (low level) 49 ERBB2 TLEEITNFL TLEEITGYLubiquitous (low level) 50 ERBB2 TVDEITGYL TLEEITGYLubiquitous (low level) 51 ERBB2 VMLGVVFGV VVLGVVFGIubiquitous (low level) 52 ERBB2 VLLGVVFGV VVLGVVFGIubiquitous (low level) 53 ERBB2 MVLGVVFGV VVLGVVFGIubiquitous (low level) 54 ERBB2 VMLGIVFGI VVLGVVFGIubiquitous (low level) 55 ERBB2 VMLGVVFGI VVLGVVFGIubiquitous (low level) 56 ERBB2 ILLGVVFGI VVLGVVFGIubiquitous (low level) 57 ERBB2 VLLGVIFGI VVLGVVFGIubiquitous (low level) 58 ERBB2 VLFGVVFGI VVLGVVFGIubiquitous (low level) 59 ERBB2 IVLGVVFGV VVLGVVFGIubiquitous (low level) 60 ERBB2 VVLGVLFGV VVLGVVFGIubiquitous (low level) 61 ERBB2 VVLGVMFGV VVLGVVFGIubiquitous (low level) 62 ERBB2 VVLGVIFGV VVLGVVFGIubiquitous (low level) 63 ERBB2 VVLGAVFGV VVLGVVFGIubiquitous (low level) 64 ERBB2 VVLGLVFGV VVLGVVFGIubiquitous (low level) 65 ERBB2 VVIGVVFGV VVLGVVFGIubiquitous (low level) 66 ERBB2 VVLGIVFGV VVLGVVFGIubiquitous (low level) 67 ERBB2 TVLGVVFGV VVLGVVFGIubiquitous (low level) 68 ERBB2 VVLGVVFGV VVLGVVFGIubiquitous (low level) 69 ERBB2 AILGVVFGI VVLGVVFGIubiquitous (low level) 70 ERBB2 AVLGVMFGI VVLGVVFGIubiquitous (low level) 71 IL13RA2 FLPFGFILV WLPFGFILI NA 72 MAGEA1KMLHYVIKV KVLEYVIKV NA 73 MAGEA3 EMNPIGHLY EVDPIGHLY NA 74 MAGEA3RVDPIGNLY EVDPIGHLY NA 75 MAGEA3 VTELVNFLL VAELVHFLL NA 76 MAGEA4HVDPATNTY EVDPASNTY NA 77 MAGEC1 KLVEWLAML KVVEFLAML NA 78 MAGEC1SLSYALLSL  SFSYTLLSL NA 79 MAGEC1 SISHTLLSL SFSYTLLSL NA 80 MAGEC1VSSFFSYVF VSSFFSYTL NA 81 MAGEC2 ALNDVEEKV ALKDVEERV NA 82 MAGEC2ALSDVEDRV ALKDVEERV NA 83 MAGEC2 ALSDAEERV ALKDVEERV NA 84 MAGEC2ATSTLMLVF ASSTLYLVF NA 85 MAGEC2 TTSTLYLVF ASSTLYLVF NA 86 SCGB2A2PLFESVISK PLLENVISK breast cancer 87 SCGB2A2 PLLETTISK PLLENVISKbreast cancer 88 MLANA ILTAILGVL ILTVILGVL melanoma 89 MLANA ILTVILGVVILTVILGVL melanoma 90 MDK ALFAVTSAV ALLALTSAV ubiquitous (low level) 91MDK ALFALTSAA ALLALTSAV ubiquitous (low level) 92 MMP2 SLPPDVQEVGLPPDVQRV ubiquitous 93 MMP2 SLPPDVQQV GLPPDVQRV ubiquitous 94 CTAG1BVAMPFATPV LAMPFATPM NA 95 ACPP ALDVYSALL ALDVYNGLL prostate cancer 96ACPP ALDMYNALL ALDVYNGLL prostate cancer 97 ACPP ALDIYNSLL ALDVYNGLLprostate cancer 98 ACPP FLFFLFFFL FLFLLFFWL prostate cancer 99 ACPPTLMSSMTNM TLMSAMTNL prostate cancer 100 STEAP1 MLAVFLPMV MIAVFLPIVpro state cancer 101 STEAP1 MLAVFLPLV MIAVFLPIV pro state cancer 102STEAP1 YLAVFLPIV MIAVFLPIV pro state cancer 103 TAG1 SLGYLFLLM SLGWLFLLLNA 104 TAG1 SLGFLFLLM SLGWLFLLL NA 105 TAG1 SLGFLFLLF SLGWLFLLL NA 106TYR MLFAVLMCL MLLAVLYCL melanoma

Those 106 antigenic peptide sequences can be further defined based onthe sequence of the reference tumor antigen, such as a tumor antigenderived from IL13RA2.

Thus, the invention relates to an immunogenic compound comprising anantigenic peptide having amino acid similarity with a tumor antigen,which antigenic peptide is selected in the group consisting of SEQ IDNO:1-106, which includes:

-   -   peptides having amino acid similarity with the tumor antigen        encoded by gene PLIN2, the said antigenic peptide being selected        in the group consisting of SEQ ID NO:1;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene ALDH1A1, the said antigenic peptide being        selected in the group consisting of SEQ ID NO:2-3;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene AFP, the said antigenic peptide being selected        in the group consisting of SEQ ID NO:4-5;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene PTPRC, the said antigenic peptide being selected        in the group consisting of SEQ ID NO:6-11;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene CEACAM5, the said antigenic peptide being        selected in the group consisting of SEQ ID NO:12-25;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene ENAH, the said antigenic peptide being selected        in the group consisting of SEQ ID NO:26;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene EZH2, the said antigenic peptide being selected        in the group consisting of SEQ ID NO:27;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene PMEL, the said antigenic peptide being selected        in the group consisting of SEQ ID NO:28;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene ERBB2, the said antigenic peptide being selected        in the group consisting of SEQ ID NO:29-70;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene IL13RA2, the said antigenic peptide being        selected in the group consisting of SEQ ID NO:71;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene MAGEA1, the said antigenic peptide being        selected in the group consisting of SEQ ID NO:72;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene MAGEA3, the said antigenic peptide being        selected in the group consisting of SEQ ID NO:73-75;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene MAGEA4, the said antigenic peptide being        selected in the group consisting of SEQ ID NO:76;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene MAGEC1, the said antigenic peptide being        selected in the group consisting of SEQ ID NO:77-80;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene MAGEC2, the said antigenic peptide being        selected in the group consisting of SEQ ID NO:81-85;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene SCGB2A2, the said antigenic peptide being        selected in the group consisting of SEQ ID NO:86-87;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene MLANA, the said antigenic peptide being selected        in the group consisting of SEQ ID NO:88-89;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene MDK, the said antigenic peptide being selected        in the group consisting of SEQ ID NO:90-91;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene MMP2, the said antigenic peptide being selected        in the group consisting of SEQ ID NO:92-93;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene CTAG1B, the said antigenic peptide being        selected in the group consisting of SEQ ID NO:94;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene ACPP, the said antigenic peptide being selected        in the group consisting of SEQ ID NO:95-99;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene STEAP1, the said antigenic peptide being        selected in the group consisting of SEQ ID NO:100-102;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene TAG1, the said antigenic peptide being selected        in the group consisting of SEQ ID NO:103-105;    -   peptides having amino acid similarity with the tumor antigen        encoded by gene TYR, the said antigenic peptide being selected        in the group consisting of SEQ ID NO:106.

Accordingly, those 106 antigenic peptides may be further categorized ina plurality of distinct families according to their reference peptide:

-   -   Family «SVASTITGV» (SEQ ID NO:107), which family includes the        amino acid sequences of SEQ ID NO: 1;    -   Family «LLYKLADLI» (SEQ ID NO:108) which family includes the        amino acid sequences of SEQ ID NO:2-3;    -   Family «QLAVSVILRV» (SEQ ID NO:109) which family includes the        amino acid sequences of SEQ ID NO:4-5;    -   Family «KFLDALISL» (SEQ ID NO:110) which family includes the        amino acid sequences of SEQ ID NO:6-11;    -   Family «GVLVGVALI» (SEQ ID NO:111) which family includes the        amino acid sequences of SEQ ID NO: 12-24;    -   Family «HLFGYSWYK» (SEQ ID NO:112) which family includes the        amino acid sequences of SEQ ID NO:25;    -   Family «TMNGSKSPV» (SEQ ID NO:113) which family includes the        amino acid sequences of SEQ ID NO:26;    -   Family «FMVEDETVL» (SEQ ID NO:114) which family includes the        amino acid sequences of SEQ ID NO:27;    -   Family «ITDQVPFSV» (SEQ ID NO:115) which family includes the        amino acid sequences of SEQ ID NO:28;    -   Family «IISAVVGIL» (SEQ ID NO:116) which family includes the        amino acid sequences of SEQ ID NO:29-40;    -   Family «ILHNGAYSL» (SEQ ID NO:117) which family includes the        amino acid sequences of SEQ ID NO:41;    -   Family «KIFGSLAFL» (SEQ ID NO:118) which family includes the        amino acid sequences of SEQ ID NO:42-43;    -   Family «PLTSIISAV» (SEQ ID NO: 119) which family includes the        amino acid sequences of SEQ ID NO:44-46;    -   Family «RLLQETELV» (SEQ ID NO:120) which family includes the        amino acid sequences of SEQ ID NO:47;    -   Family «TLEEITGYL» (SEQ ID NO:121) which family includes the        amino acid sequences of SEQ ID NO:48-50;    -   Family «VVLGVVFGI» (SEQ ID NO:122) which family includes the        amino acid sequences of SEQ ID NO:51-70;    -   Family «WLPFGFILI» (SEQ ID NO:123) including sequence SEQ ID        NO:71;    -   Family «KVLEYVIKV» (SEQ ID NO:124) which family includes the        amino acid sequences of SEQ ID NO:72;    -   Family «EVDPIGHLY» (SEQ ID NO:125) which family includes the        amino acid sequences of SEQ ID NO:73-74;    -   Family «VAELVHFLL» (SEQ ID NO: 126) which family includes the        amino acid sequences of SEQ ID NO:75;    -   Family «EVDPASNTY» (SEQ ID NO:127) which family includes the        amino acid sequences of SEQ ID NO:76;    -   Family «KVVEFLAML» (SEQ ID NO:128) which family includes the        amino acid sequences of SEQ ID NO:77;    -   Family «SFSYTLLSL» (SEQ ID NO:129) which family includes the        amino acid sequences of SEQ ID NO:78-79;    -   Family «VSSFFSYTL» (SEQ ID NO:130) which family includes the        amino acid sequences of SEQ ID NO:80;    -   Family «ALKDVEERV» (SEQ ID NO:131) which family includes the        amino acid sequences of SEQ ID NO:81-83;    -   Family «ASSTLYLVF» (SEQ ID NO:132) which family includes the        amino acid sequences of SEQ ID NO:84-85;    -   Family «PLLENVISK» (SEQ ID NO:133) which family includes the        amino acid sequences of SEQ ID NO:86-87;    -   Family «ILTVILGVL» (SEQ ID NO:134) which family includes the        amino acid sequences of SEQ ID NO:88-89;    -   Family «ALLALTSAV» (SEQ ID NO:135) which family includes the        amino acid sequences of SEQ ID NO:90-91;    -   Family «GLPPDVQRV» (SEQ ID NO: 136) which family includes the        amino acid sequences of SEQ ID NO:92-93;    -   Family «LAMPFATPM» (SEQ ID NO:137) which family includes the        amino acid sequences of SEQ ID NO:94;    -   Family «ALDVYNGLL» (SEQ ID NO:138) which family includes the        amino acid sequences of SEQ ID NO:95-97;    -   Family «FLFLLFFWL» (SEQ ID NO:139) which family includes the        amino acid sequences of SEQ ID NO:98;    -   Family «TLMSAMTNL» (SEQ ID NO:140) which family includes the        amino acid sequences of SEQ ID NO:99;    -   Family «MIAVFLPIV» (SEQ ID NO:141) which family includes the        amino acid sequences of SEQ ID NO:100-102;    -   Family «SLGWLFLLL» (SEQ ID NO:142) which family includes the        amino acid sequences of SEQ ID NO: 103-105;    -   Family «MLLAVLYCL» (SEQ ID NO:143) which family includes the        amino acid sequences of SEQ ID NO: 106.

According to a preferred embodiment, an antigenic peptide of theinvention is selected from the group consisting of peptides orpolypeptides comprising, or consisting of, the amino acid sequence SEQID NO:71.

According to an exemplified embodiment, the antigenic peptide of theinvention is a peptide or polypeptide comprising, or consisting of, theamino acid sequence of SEQ ID NO:71.

More preferably, the antigenic peptide of the invention is selected fromthe group consisting of peptides or polypeptides comprising orconsisting of an amino acid sequence according to any one of SEQ ID NOs:17, 31, 32, 51, 52, 55, 56, 59, 68, 89, 94, 100, 101 or 102. It is alsomore preferred that the antigenic peptide of the invention is selectedfrom the group consisting of peptides or polypeptides comprising orconsisting of an amino acid sequence according to any one of SEQ ID NOs:26, 28, 47, 51, 52, 55, 56, 77, 93, 101 or 102. Even more preferably,the antigenic peptide of the invention is selected from the groupconsisting of peptides or polypeptides comprising or consisting of anamino acid sequence according to any one of SEQ ID NOs: 51, 52, 55, 56,101 or 102. Still more preferably, the antigenic peptide of theinvention is selected from the group consisting of peptides orpolypeptides comprising or consisting of an amino acid sequenceaccording to any one of SEQ ID NOs: 51, 52, 55 or 56. It is also stillmore preferred that the antigenic peptide of the invention is selectedfrom the group consisting of peptides or polypeptides comprising orconsisting of an amino acid sequence according to any one of SEQ ID NOs:101 or 102.

According to some embodiments, the immunogenic compound comprises, orconsists of, an antigenic peptide of formula (I)PepNt-CORE-PepCt  (I), wherein:

-   -   “PepNt” consists of a polypeptide having an amino acid length        varying from 0 to 500 amino acid residues and located at the        N-terminal end of the polypeptide of formula (I);    -   CORE consists of a polypeptide comprising, or alternatively        consisting of, an amino acid sequence selected from the group        consisting of SEQ ID NO: 1 to 106 (which includes SEQ ID NO:        71), in particular an amino acid sequence as set forth in any        one of SEQ ID NOs 26, 28, 47, 51, 52, 55, 56, 77, 93, 101 or 102        or an amino acid sequence as set forth in any one of 17, 31, 32,        51, 52, 55, 56, 59, 68, 89, 94, 100, 101 or 102, such as an        amino acid sequence as set forth in any one of SEQ ID NOs 51,        52, 55, 56, 101 or 102; and    -   “PepCt” consists of a polypeptide having an amino acid length        varying from 0 to 500 amino acid residues and located at the        C-terminal end of the polypeptide of formula (I).

According to one particular embodiment, the immunogenic compoundcomprises or consists of an antigenic peptide of formula (Ia) or (Ib)PepNt-CORE  (Ia); orCORE-PepCt  (Ib).wherein “PepNt” and “PepCt” and CORE are as defined above.

According to some even more particular embodiments, the antigenicpeptide or immunogenic above, as defined above, comprises from 9 to 1000amino acids; which includes 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67? 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1000amino acids.

According to said embodiment, the length of “PepNt” and “PepCt”, ifapplicable, are defined accordingly.

Thus, “PepNt” and “PepCt”, as defined above, may comprise from 0 to 500amino acid residues; which includes 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400,450, and 500 amino acid residues.

The types of carrier molecules used for generating an immunogeniccompound of the invention, such as the ones comprising or consisting ofa peptide of formula (I) linked to a carrier molecule, are well in thegeneral knowledge of the one skilled in the art. The function of thecarrier molecule is to provide cytokine help (or T-cell help) in orderto enhance the immune response against tumor antigen.

Preferably, the antigenic peptide is linked to a carrier molecule, inparticular to a carrier protein, preferably by covalent or non-covalentbond. The carrier molecule to which the peptide is optionally bound canbe selected from a wide variety of known carriers. Examples of carriermolecules for vaccine purposes encompass proteins such as human orbovine serum albumin and keyhole limpet haemocyanin (KLH) and fattyacids. Other embodiments of carrier molecules to which an antigenicpeptide of formula (I) may be covalently linked include bacterial toxinsor toxoids, such as diphtheria, cholera, E. coli heat labile or tetanustoxoids, the N. meningitidis outer membrane protein (European patentapplication no EP0372501), synthetic peptides (European patentapplications n° EP0378881 and no EP0427347), heat shock proteins (PCTapplication no WO93/17712), Pertussis proteins (PCT application noWO98/58668), protein D from H. influenzae (PCT application noWO00/56360) and toxin A or B from C. difficile (International patentapplication WO00/61761).

According to one embodiment, the carrier protein or carrier peptide is aHHD-DR3 carrier peptide MAKTIAYDEEARRGLERGLN (SEQ ID NO:144).

According to one embodiment, “PepNt” and/or “PepCt” may correspond to acarrier protein or carrier peptide, such as the HHD-DR3 carrier peptideMAKTIAYDEEARRGLERGLN (SEQ ID NO:144).

According to one embodiment, the immunogenic compound comprises orconsists of the carrier peptide of sequence SEQ ID NO:144 linkedcovalently to the N-terminus of the antigenic peptide of sequence SEQ IDNO:71.

More preferably, the carrier protein or carrier peptide is aprotein/peptide having immuno-adjuvant properties, such as providingstimulation of CD4+ Th1 cells as described herein. A preferred examplethereof is a non-tumor antigen that recalls immune memory or provides anon-specific help or could be a specific tumor-derived helper peptide,such as tetanus helper peptide, keyhole limpet hemocyanin peptide orPADRE peptide. Another preferred example is a specific tumor derivedhelper peptide, which may be presented by MHC II, in particular byHLA-DR, HLA-DP or HLA-DQ, such as fragments of shared overexpressedtumor antigens, e.g. HER2, NY-ESO-1, hTERT or IL13RA2, as describedabove.

Accordingly, “PepNt” and/or “PepCt” may preferably correspond to such aprotein/peptide having immuno-adjuvant properties, such as providingstimulation of CD4+ Th1 cells as described herein.

Moreover, the immunogenic compound comprises or consists of such aprotein/peptide having immuno-adjuvant properties, such as providingstimulation of CD4+ Th1 cells as described herein, linked covalently tothe N-terminus of the antigenic peptide having an amino acid sequenceselected from the group consisting of SEQ ID NO: 1 to 106 (whichincludes SEQ ID NO: 71), in particular an amino acid sequence as setforth in any one of SEQ ID NOs 26, 28, 47, 51, 52, 55, 56, 77, 93, 101or 102 or an amino acid sequence as set forth in any one of 17, 31, 32,51, 52, 55, 56, 59, 68, 89, 94, 100, 101 or 102, such as an amino acidsequence as set forth in any one of SEQ ID NOs: 51, 52, 55, 56, 101 or102.

According to one embodiment, the said antigenic peptide is covalentlybound to the carrier molecule through a linker moiety.

The said restricted family of linker agents encompasses, or evenconsists of, the linker agents named GMBS, sulfo-GMBS, SMPB andsulfo-SMPB.

In some embodiments of an immunogenic compound as defined above, thesaid linker agent is selected form the group consisting of GMBS(N-[γ-maleimidobutyryl-oxy]succinimide ester), Sulfo-GMBS(N-[γ-maleimidobutyryl-oxy]sulfosuccinimide ester), SMPB (succinimidyl4-[p-maleimidophenyl]butyrate) and Sulfo-SMPB (sulfosuccinimidyl4-[p-maleimidophenyl]butyrate).

Methods for conjugating two proteins with a linker agent in general, andmore particularly with a linker agent selected from the group consistingof GMBS, Sulfo-GMBS, SMPB and Sulfo-SMPB, are well known by the oneskilled in the art. Illustratively, such protocols are disclosed in theleaflets that are made publicly available by the Pierce Company(Illinois, USA). GMBS, Sulfo-GMBS, SMPB and Sulfo-SMPB consist ofheterobifunctional linker agents that contain both aN-hydroxysuccinimide (NHS) ester group and a maleimide group.Conjugation using GMBS, Sulfo-GMBS, SMPB or Sulfo-SMPB is usuallyperformed by a two-step procedure. In a first step, the amine-containingprotein is reacted with a several-fold molar excess of the linker agentat pH 7-9 to form amide bonds, followed by removal of excess non-reactedlinker agent, usually by desalting or dialysis. In a second step, thesulfhydryl-containing molecule (e.g. peptide of formula (I)) is added toreact with the maleimide groups already attached to the first protein atpH 6.5-7.5 to form stable thioether bonds.

Using SMPB or Sulfo-SMPB as linker agents for covalently linkingpeptides of formula (I) to the amine-containing carrier protein, leadsto a conjugate of formula (II) below:

wherein:

-   -   R1 consists of one reactive group of the amine-containing        carrier protein, and wherein the NH group attached thereto        derives from (i) the alpha amino group located at the N-terminal        end of the amine-containing carrier protein or (ii) a lateral        chain amino group from a Lysine (K) amino acid residue of the        amine-containing carrier protein.    -   R2 consists of a peptide of formula (I), and wherein the        sulphur (S) atom attached thereto derives from a sulfhydryl (SH)        group of a cysteine residue located at the N-terminal end or at        the C-terminal end of a peptide of formula (I). In some        embodiments, the sulfhydryl moiety could be part of an unnatural        amino acid, or any other molecule present at the end of the        peptide of formula (I).

Using GMBS or Sulfo-GMBS as linker agents for covalently linkingpeptides of formula (I) to the amine-containing carrier protein, inparticular the CRM197 carrier, protein leads to a conjugate of formula(III) below:

wherein:

-   -   R1 consists of one reactive group of the amine-containing        carrier protein, and wherein the NH group attached thereto        derives from (i) the alpha amino group located at the N-terminal        end of the amine-containing carrier proteinor (ii) a lateral        chain amino group from a Lysine (K) amino acid residue of the        amine-containing carrier protein.    -   R2 consists of a peptide of formula (I), and wherein the        sulphur (S) atom attached thereto derives from a sulfhydryl (SH)        group of a cysteine residue located at the N-terminal end or at        the C-terminal end of a peptide of formula (I). In some        embodiments, the sulfhydryl moiety could be part of an unnatural        amino acid, or any other molecule present at the end of the        peptide of formula (I).

In a further aspect the present invention provides a cell loaded with atleast one immunogenic compound according to the present invention orwith at least one antigenic peptide according to the present invention.A preferred antigenic peptide is a peptide or polypeptide having anamino acid sequence as set forth in in any one of SEQ ID NOs 26, 28, 47,51, 52, 55, 56, 77, 93, 101 or 102 or an amino acid sequence as setforth in any one of 17, 31, 32, 51, 52, 55, 56, 59, 68, 89, 94, 100, 101or 102, such as an amino acid sequence as set forth in any one of SEQ IDNOs 51, 52, 55, 56, 101 or 102. Also combinations thereof are preferred,namely, cells loaded with distinct antigenic peptides according to thepresent invention (or with the respective immunogenic compound(s)).

A preferred cell is an antigen presenting cell (APC), more preferably adendritic cell (DC).

Antigen-presenting cells (APCs) are of particular interest, as theirmain function is to process antigens and present it on the cell surfaceto the T cells of the immune system, so as to initiate and modulateT-cell responses in vivo. In the context of the present invention, it ispreferred that the APCs are loaded with the antigenic peptide(s) and/orimmunogenic compound(s) according to the invention, which can be done byexposing APCs in vitro with said antigenic peptide(s) and/or immunogeniccompound(s) (Rizzo M M, Alaniz L, Mazzolini G. Ex vivo loading ofautologous dendritic cells with tumor antigens. Methods Mol Biol. 2014;1139:41-4; Rolinski J, Hus I. Breaking immunotolerance of tumors: a newperspective for dendritic cell therapy. J Immunotoxicol. 2014 October;11(4):311-8).

Preferred antigen-presenting cells according to the invention aredendritic cells (DCs). It can indeed be advantageous to combine at leastone antigenic peptide or immunogenic compound according to the inventionwith dendritic cells, as those are the most potent antigen-presentingcells and have been reported to be frequently functionally defective incancer patients. Dendritic cells can be easily obtained by the skilledperson in the art from either healthy compatible donors (i.e. thedendritic cells are HLA-related) or from the patient himself providedthat they are functional (i.e. the dendritic cells are autologous), forexample by direct isolation from the peripheral blood, or by derivationfrom peripheral blood cells such as CD14+ monocytes or CD34+hematopoietic precursors (Figdor C G, de Vries I J, Lesterhuis W J,Melief C J. Dendritic cell immunotherapy: mapping the way. Nat Med. 2004May; 10(5):475-80). Dendritic cells can indeed be distinguished fromother cells of peripheral blood by their surface markers, such as S100,p55, CD83, and/or OX62, and may thus be isolated and purified based onsaid markers using cell cultures techniques well-known in the art.

In a further aspect, the present invention provides a nucleic acidencoding an antigenic peptide according to the present invention or animmunogenic compound according to the present invention, wherein theimmunogenic compound is a peptide or a protein. Preferably, theantigenic peptide is a peptide or polypeptide having an amino acidsequence as set forth in in any one of SEQ ID NOs: 26, 28, 47, 51, 52,55, 56, 77, 93, 101 or 102 or an amino acid sequence as set forth in anyone of 17, 31, 32, 51, 52, 55, 56, 59, 68, 89, 94, 100, 101 or 102, suchas an amino acid sequence as set forth in any one of SEQ ID NOs: 51, 52,55, 56, 101 or 102; and/or an antigenic peptide of formula (I) asdescribed above.

Nucleic acids preferably comprise single stranded, double stranded orpartially double stranded nucleic acids, preferably selected fromgenomic DNA, cDNA, RNA, antisense DNA, antisense RNA, complementaryRNA/DNA sequences with or without expression elements, a mini-gene, genefragments, regulatory elements, promoters, and combinations thereof.Further preferred examples of nucleic acid (molecules) and/orpolynucleotides include, e.g., a recombinant polynucleotide, a vector,an oligonucleotide, an RNA molecule such as an rRNA, an mRNA, or a tRNA,or a DNA molecule as described above. It is thus preferred that thenucleic acid (molecule) is a DNA molecule or an RNA molecule; preferablyselected from genomic DNA; cDNA; rRNA; mRNA; antisense DNA; antisenseRNA; complementary RNA and/or DNA sequences; RNA and/or DNA sequenceswith or without expression elements, regulatory elements, and/orpromoters; a vector; and combinations thereof.

Accordingly, the nucleic acid molecule may be a vector. The term“vector”, as used in the context of the present invention, refers to anucleic acid molecule, preferably to an artificial nucleic acidmolecule, i.e. a nucleic acid molecule which does not occur in nature. Avector in the context of the present invention is suitable forincorporating or harboring a desired nucleic acid sequence. Such vectorsmay be storage vectors, expression vectors, cloning vectors, transfervectors etc. A storage vector is a vector which allows the convenientstorage of a nucleic acid molecule. Thus, the vector may comprise asequence corresponding, e.g., to a desired antigenic peptide accordingto the present invention. An expression vector may be used forproduction of expression products such as RNA, e.g. mRNA, or peptides,polypeptides or proteins. For example, an expression vector may comprisesequences needed for transcription of a sequence stretch of the vector,such as a promoter sequence. A cloning vector is typically a vector thatcontains a cloning site, which may be used to incorporate nucleic acidsequences into the vector. A cloning vector may be, e.g., a plasmidvector or a bacteriophage vector. A transfer vector may be a vector thatis suitable for transferring nucleic acid molecules into cells ororganisms, for example, viral vectors. A vector in the context of thepresent invention may be, e.g., an RNA vector or a DNA vector.Preferably, a vector is a DNA molecule. For example, a vector in thesense of the present application comprises a cloning site, a selectionmarker, such as an antibiotic resistance factor, and a sequence suitablefor multiplication of the vector, such as an origin of replication.Preferably, a vector in the context of the present application is aplasmid vector. Preferably, a vector in the context of the presentapplication is an expression vector. A preferred vector is a vector forexpression in bacterial cells. More preferably, the vector is useful forexpression in so-called “live bacterial vaccine vectors”, wherein livebacterial cells (such as bacteria or bacterial spores, e.g., endospores,exospores or microbial cysts) can serve as vaccines. Preferred examplesthereof are described in da Silva et al., Live bacterial vaccinevectors: an overview; Braz J Microbiol. 2015 Mar. 4; 45(4):1117-29.

Nucleic acids encoding antigenic peptides according to the invention maybe in the form of naked nucleic acids, or nucleic acids cloned intoplasmids or viral vectors (Tregoning and Kinnear, Using Plasmids as DNAVaccines for Infectious Diseases. Microbiol Spectr. 2014 December; 2(6).doi: 10.1128/microbiolspec.PLAS-0028-2014), the latter beingparticularly preferred. Examples of suitable viral vectors according tothe invention include, without limitation, retrovirus, adenovirus,adeno-associated virus (AAV), herpes virus and poxvirus vectors. It iswithin the skill of the person in the art to clone a nucleic acid into aplasmid or viral vector, using standard recombinant techniques in theart.

In a further aspect, the present invention also provides a host cellcomprising the nucleic acid according to the present invention, whereinthe nucleic acid is preferably a vector. Preferably, the host cell is abacterial cell. Such a host cell may be preferably used for productionof the antigenic peptide according to the present invention or theimmunogenic compound according to the present invention. Moreover, sucha host cell may also be an active component in a vaccine.

Preferably, the host cell is a bacterial cell, preferably a gutbacterial cell. Such a bacterial host cell may serve as “live bacterialvaccine vector”, wherein live bacterial cells (such as bacteria orbacterial spores, e.g., endospores, exospores or microbial cysts) canserve as vaccines. Preferred examples thereof are described in da Silvaet al., Live bacterial vaccine vectors: an overview; Braz J Microbiol.2015 Mar. 4; 45(4):1117-29.

Bacterial cells (such as bacteria or bacterial spores, e.g., endospores,exospores or microbial cysts), in particular (entire) gut bacterialspecies, can be advantageous, as they have the potential to trigger agreater immune response than the (poly)peptides or nucleic acids theycontain.

Alternatively, bacterial cells, in particular gut bacteria, according tothe invention may be in the form of probiotics, i.e. of live gutbacterium, which can thus be used as food additive due to the healthbenefits it can provide. Those can be, for example, lyophilized ingranules, pills or capsules, or directly mixed with dairy products forconsumption.

In a further aspect, the present invention provides a nanoparticleloaded with

-   -   at least one of the immunogenic compounds according to the        present invention, or    -   at least one of the antigenic peptides according to the present        invention;

and, optionally, with an adjuvant.

Nanoparticles, in particular for use as vaccines, are known in the artand described, for example, in Shao et al., Nanoparticle-basedimmunotherapy for cancer, ACS Nano 2015, 9(1):16-30; Zhao et al.,Nanoparticle vaccines, Vaccine 2014, 32(3):327-37; and Gregory et al.,Vaccine delivery using nanoparticles, Front Cell Infect Microbiol. 2013,3:13, doi: 10.3389/fcimb.2013.00013. eCollection 2013, Review. Inparticular, the nanoparticle is used for delivery of the antigenicpeptide (or the polypeptide/protein/nucleic acid comprising theantigenic peptide) and may optionally also act as an adjuvant. Theantigenic peptide (the polypeptide/protein/nucleic acid comprising theantigenic peptide) is typically either encapsulated within thenanoparticle or linked/bound to (decorated onto) the surface of thenanoparticle (“coating”). Compared to conventional approaches,nanoparticles can protect the payload (antigen/adjuvant) from thesurrounding biological milieu, increase the half-life, minimize thesystemic toxicity, promote the delivery to APCs, or even directlytrigger the activation of TAA-specific T-cells. Preferably, thenanoparticle has a size (diameter) of no more than 300 nm, morepreferably of no more than 200 nm and most preferably of no more than100 nm. Such nanoparticles are adequately sheltered from phagocyteuptake, with high structural integrity in the circulation and longcirculation times, capable of accumulating at sites of tumor growth, andable to penetrate deep into the tumor mass.

Examples of nanoparticles include polymeric nanoparticles such aspoly(ethylene glycol) (PEG) and poly (D,L-lactic-coglycolic acid)(PLGA); inorganic nanoparticles such as gold nanoparticles, iron oxidebeads, iron-oxide zinc-oxide nanoparticles, carbon nanotubes andmesoporous silica nanoparticles; liposomes, such as cationic liposomes;immunostimulating complexes (ISCOM); virus-like particles (VLP); andself-assembled proteins.

Polymeric nanoparticles are nanoparticles based on/comprising polymers,such as poly(d,l-lactide-co-glycolide) (PLG), poly(d,l-lactic-coglycolicacid)(PLGA), poly(g-glutamic acid) (g-PGA), poly(ethylene glycol) (PEG),and polystyrene. Polymeric nanoparticles may entrap an antigen (e.g.,the antigenic peptide or a (poly)peptide comprising the same) or bindto/conjugate to an antigen (e.g., the antigenic peptide or a(poly)peptide comprising the same). Polymeric nanoparticles may be usedfor delivery, e.g. to certain cells, or sustain antigen release byvirtue of their slow biodegradation rate. For example, g-PGAnanoparticles may be used to encapsulate hydrophobic antigens.Polystyrene nanoparticles can conjugate to a variety of antigens as theycan be surface-modified with various functional groups. Polymers, suchas Poly(L-lactic acid) (PLA), PLGA, PEG, and natural polymers such aspolysaccharides may also be used to synthesize hydrogel nanoparticles,which are a type of nano-sized hydrophilic three-dimensional polymernetwork. Nanogels have favorable properties including flexible meshsize, large surface area for multivalent conjugation, high watercontent, and high loading capacity for antigens. Accordingly, apreferred nanoparticle is a nanogel, such as a chitosan nanogel.Preferred polymeric nanoparticles are nanoparticles based on/comprisingpoly(ethylene glycol) (PEG) and poly (D,L-lactic-coglycolic acid)(PLGA).

Inorganic nanoparticles are nanoparticles based on/comprising inorganicsubstances, and examples of such nanoparticles include goldnanoparticles, iron oxide beads, iron-oxide zinc-oxide nanoparticles,carbon nanoparticles (e.g., carbon nanotubes) and mesoporous silicananoparticles. Inorganic nanoparticles provide a rigid structure andcontrollable synthesis. For example, gold nanoparticles can be easilyproduced in different shapes, such as spheres, rods, cubes. Inorganicnanoparticles may be surface-modified, e.g. with carbohydrates. Carbonnanoparticles provide good biocompatibility and may be produced, forexample, as nanotubes or (mesoporous) spheres. For example, multiplecopies of the antigenic peptide according to the present invention (or a(poly)peptide comprising the same) may be conjugated onto carbonnanoparticles, e.g. carbon nanotubes. Mesoporous carbon nanoparticlesare preferred for oral administration. Silica-based nanoparticles(SiNPs) are also preferred. SiNPs are biocompatible and show excellentproperties in selective tumor targeting and vaccine delivery. Theabundant silanol groups on the surface of SiNPs may be used for furthermodification to introduce additional functionality, such as cellrecognition, absorption of specific biomolecules, improvement ofinteraction with cells, and enhancement of cellular uptake. Mesoporoussilica nanoparticles are particularly preferred.

Liposomes are typically formed by phospholipids, such as1,2-dioleoyl-3-trimethylammonium propane (DOTAP). In general, cationicliposomes are preferred. Liposomes are self-assembling with aphospholipid bilayer shell and an aqueous core. Liposomes can begenerated as unilameller vesicles (having a single phospholipid bilayer)or as multilameller vesicles (having several concentric phospholipidshells separated by layers of water). Accordingly, antigens can beencapsulated in the core or between different layers/shells. Preferredliposome systems are those approved for human use, such as Inflexal® Vand Epaxal®.

Immunostimulating complexes (ISCOM) are cage like particles of about 40nm (diameter), which are colloidal saponin containing micelles, forexample made of the saponin adjuvant Quil A, cholesterol, phospholipids,and the (poly)peptide antigen (such as the antigenic peptide or apolypeptide comprising the same). These spherical particles can trap theantigen by apolar interactions. Two types of ISCOMs have been described,both of which consist of cholesterol, phospholipid (typically eitherphosphatidylethanolamine or phosphatidylcholine) and saponin (such asQuilA).

Virus-like particles (VLP) are self-assembling nanoparticles formed byself-assembly of biocompatible capsid proteins. Due to thenaturally-optimized nanoparticle size and repetitive structural orderVLPs can induce potent immune responses. VLPs can be derived from avariety of viruses with sizes ranging from 20 nm to 800 nm, typically inthe range of 20-150 nm. VLPs can be engineered to express additionalpeptides or proteins either by fusing these peptides/proteins to theparticle or by expressing multiple antigens. Moreover, antigens can bechemically coupled onto the viral surface to produce bioconjugate VLPs.

Examples of self-assembled proteins include ferritin and major vaultprotein (MVP). Ferritin is a protein that can self-assemble intonearly-spherical 10 nm structure. Ninety-six units of MVP canself-assemble into a barrel-shaped vault nanoparticle, with a size ofapproximately 40 nm wide and 70 nm long. Antigens that are geneticallyfused with a minimal interaction domain can be packaged inside vaultnanoparticles by self-assembling process when mixed with MVPs.Accordingly, the antigen (such as the antigenic peptide according to thepresent invention of a polypeptide comprising the same) may be fused toa self-assembling protein or to a fragment/domain thereof, such as theminimal interaction domain of MVP. Accordingly, the present inventionalso provides a fusion protein comprising a self-assembling protein (ora fragment/domain thereof) and the antigenic peptide according to thepresent invention.

In general, preferred examples of nanoparticles (NPs) include iron oxidebeads, polystyrene microspheres, poly(γ-glutamic acid) (γ-PGA) NPs, ironoxide-zinc oxide NPs, cationized gelatin NPs, pluronic-stabilizedpoly(propylene sulfide) (PPS) NPs, PLGA NPs, (cationic) liposomes,(pH-responsive) polymeric micelles, PLGA, cancer cell membrane coatedPLGA, lipid-calcium-phosphate (LCP) NPs, liposome-protamine-hyaluronicacid (LPH) NPs, polystyrene latex beads, magnetic beads, iron-dextranparticles and quantum dot nanocrystals.

Preferably, the nanoparticle further comprises an adjuvant, for examplea toll-like receptor (TLR) agonist. Thereby, the antigenic peptide (thepolypeptide/protein/nucleic acid comprising the antigenic peptide) canbe delivered together with an adjuvant, for example toantigen-presenting cells (APCs), such as dendritic cells (DCs). Theadjuvant may be encapsulated by the nanoparticle or bound to/conjugatedto the surface of the nanoparticle, preferably similarly to theantigenic peptide.

Particularly preferred adjuvants are polyinosinic:polycytidylic acid(also referred to as “poly I:C”) and/or its derivative poly-ICLC. PolyI:C is a mismatched double-stranded RNA with one strand being a polymerof inosinic acid, the other a polymer of cytidylic acid. Poly I:C is animmunostimulant known to interact with toll-like receptor 3 (TLR3). PolyI:C is structurally similar to double-stranded RNA, which is the“natural” stimulant of TLR3. Accordingly, poly I:C may be considered asynthetic analog of double-stranded RNA. Poly-ICLC is a syntheticcomplex of carboxymethylcellulose, polyinosinic-polycytidylic acid, andpoly-L-lysine double-stranded RNA. Similar to poly I:C, also poly-ICLCis a ligand for TLR3. Poly I:C and poly-ICLC typically stimulate therelease of cytotoxic cytokines. A preferred example of poly-ICLC isHiltonol®.

Immunogenic Compositions and Kits

Immunogenic compositions according to the invention comprises at leastone of the following:

-   -   an antigenic peptide according to the present invention,    -   an immunogenic compound according to the present invention,    -   a nanoparticle according to the present invention,    -   a cell according to the present invention,    -   a nucleic acid according to the present invention, or    -   a host cell according to the present invention.

Preferably, the immunogenic composition further comprises one or morepharmaceutically acceptable excipients or carriers.

The immunogenic composition of the invention may be in any form suitablefor the purposes of the invention. For example, said composition may bein a form suitable for parenteral, enteral or topical administration,such as a liquid suspension, a solid dosage form (granules, pills,capsules or tablets), or a paste or gel. It is within the skill of theperson in the art to select the appropriate form of the composition forthe intended purpose.

Indeed, in the context of the present invention, it can be particularlyadvantageous to use (poly)peptides, or nucleic acids encoding thereof,because of their ease of manufacturing at a low cost and relative safetywith no potential for reassortment, infection or recombination.

Antigenic peptides of the invention may be administered in the form ofimmunogenic compounds according to the present invention, cells loadedtherewith according to the present invention, nanoparticles according tothe present invention, nucleic acids according to the present invention,host cells according to the present invention and/or immunogeniccompositions according to the present invention.

According to one embodiment, they may be administered in the form of amicro-organism such as a gut bacterial species.

Entire gut bacterial species can also be advantageous as they have thepotential to trigger a greater immune response than the (poly)peptidesor nucleic acids they contain.

Alternatively, gut bacteria according to the invention may be in theform of probiotics, i.e. of live gut bacterium, which can thus be usedas food additive thanks to the health benefits it can provide. Those canbe for example lyophilized in granules, pills or capsules, or directlymixed with dairy products for consumption.

One skilled in the art would readily understand that an antigenicpeptide of the invention can be selected based upon the nature of thecancer to be prevented or treated, and/or on the human gene/human tumorantigen involved in said cancer. For example, should one wish to preventor treat melanoma which involves a Glycoprotein 100 (gp100), a TRP1, aTRP2, a tyrosinase and/or a Melan A/MART1 antigen, one can select any ofthe corresponding antigenic peptide(s) as described in Table 1A.

It shall be understood that co-administration of several antigenicpeptides of the invention is particularly preferred, so as to enhancethe immune response.

Thus, according to a preferred embodiment, the composition of theinvention comprises at least 2 antigenic peptides (which may be in theform of immunogenic compounds) as defined above, which includes at least3 antigenic peptides, or at least 4 antigenic peptides, or at least 5antigenic peptides, or at least 6 antigenic peptides, or at least 7antigenic peptides, or at least 8 antigenic peptides, or at least 9antigenic peptides, or at least 10 antigenic peptides, or at least 11antigenic peptides, or at least 12 antigenic peptides, or at least 13antigenic peptides, or at least 14 antigenic peptides, or at least 15antigenic peptides, or at least 20 antigenic peptides, or at least 25antigenic peptides, or at least 50 antigenic peptides, or at least 100antigenic peptides, or at least 500 antigenic peptides, or at least 1000antigenic peptides, or at least 1500 antigenic peptides. It is withinthe skill of the person in the art to select the combination ofantigenic peptides and/or immunogenic compounds that is suitable for theintended purpose. For example, should one wish to prevent or treatmelanoma which involves a tumor antigen encoded by a gene according toTable 1B, one can select any combination of the corresponding antigenicpeptides as described in Table 1A.

In a particularly preferred embodiment two distinct antigenic peptidesaccording to the present invention (e.g., relating to the same type ofcancer and/or to the same reference antigen) are combined. In otherwords, the composition according to the present invention preferablycomprises

(i) two distinct immunogenic compounds according to the presentinvention;

(ii) two distinct antigenic peptides according to the present invention;

(iii) two distinct nanoparticles according to the present invention; or

(iv) two distinct nucleic acids according to the present invention.

The composition according to the invention can further comprise otheractive agents, for example such, which can enhance the effects of theantigenic peptide or immunogenic compound. Alternatively, thecomposition may not comprise any other active agents (i.e., other thanthe antigenic peptide according to the present invention, theimmunogenic compound according to the present invention, thenanoparticle according to the present invention, the cell according tothe present invention, the nucleic acid according to the presentinvention, or the host cell according to the present invention).

According to a preferred embodiment, said composition further comprisesat least one immunostimulatory agent, in particular so as to potentiatethe immune response mediated by the antigenic peptide. Preferredimmunostimulatory agents according to the invention include, withoutlimitation, immune adjuvants, antigen-presenting cells, and combinationsthereof. Preferably, the immunostimulatory agent is an immune adjuvantor an antigen-presenting cell (APC).

Some immune adjuvants are indeed capable of favoring and prolonging theduration of interaction between an antigen and the immune system, whileothers are capable of recruiting and activating cells of the naturalimmunity so as to induce an adaptive response. The adjuvants belongingto the former category include, without limitation, mineral compoundssuch as alum, aluminum hydroxide, aluminum phosphate, calcium phosphatehydroxide; and oil-based emulsions such as paraffin oil, starch oil,Freund's complete/incomplete adjuvant (FCA/FIA), saponins (e.g. from theplants Quillaja, Soybean, Polygala senega). The adjuvants of belongingto the latter category include, without limitation, immunostimulatorycomplexes (ISCOMs) such as cytokines (e.g. GM-CSF; Interleukins such asIL-1, IL-2, IL6, IL8, or IL12; Tumor necrosis factors (TNFs) such asTNFα or TNFβ; Interferons IFNS such as IFNα, IFNβ, IFNγ or IFNδ, etc);ligands of toll-like receptors (TLRs) such as imiquimod, resiquimod orMPL; exosomes such as exosomes derived from dendritic cells (DCs) orfrom tumor cells; bacterial products such as heat-shock proteins (HSPssuch as gp96, hsp90, hsp70, calreticulin, hsp110, hsp170),pathogen-associated molecular patterns (PAMPs), trehalose dimicolate(TDM), muramyldipeptide (MDP), polysaccharide (PLS) such aspolysaccharide-K.

According to one embodiment, the immune adjuvant may be the HHD-DR3peptide MAKTIAYDEEARRGLERGLN (SEQ ID NO:144).

More preferably, the immune adjuvants is a protein/peptide havingimmuno-adjuvant properties, such as providing stimulation of CD4+ Th1cells, as described herein. A preferred example thereof is a non-tumorantigen that recalls immune memory or provides a non-specific help orcould be a specific tumor-derived helper peptide, such as tetanus helperpeptide, keyhole limpet hemocyanin peptide or PADRE peptide, asdescribed herein. Another preferred example is a specific tumor derivedhelper peptide, which may be presented by MHC II, in particular byHLA-DR, HLA-DP or HLA-DQ, such as fragments of shared overexpressedtumor antigens, e.g. HER2, NY-ESO-1, hTERT or IL13RA2, as describedabove.

Particularly preferred adjuvants are polyinosinic:polycytidylic acid(also referred to as “poly I:C”) and/or its derivative poly-ICLC. PolyI:C is a mismatched double-stranded RNA with one strand being a polymerof inosinic acid, the other a polymer of cytidylic acid. Poly I:C is animmunostimulant known to interact with toll-like receptor 3 (TLR3). PolyI:C is structurally similar to double-stranded RNA, which is the“natural” stimulant of TLR3. Accordingly, poly I:C may be considered asynthetic analog of double-stranded RNA. Poly-ICLC is a syntheticcomplex of carboxymethylcellulose, polyinosinic-polycytidylic acid, andpoly-L-lysine double-stranded RNA. Similar to poly I:C, also poly-ICLCis a ligand for TLR3. Poly I:C and poly-ICLC typically stimulate therelease of cytotoxic cytokines. A preferred example of poly-ICLC isHiltonol®.

Antigen-presenting cells (APCs) are also of particular interest, astheir main function is to process antigens and present it on the cellsurface to the T cells of the immune system, so as to initiate andmodulate T-cell responses in vivo. In the present composition, it ispreferred that the APCs are loaded with the antigenic peptide(s) and/orimmunogenic compound(s) according to the invention, which can be done byexposing APCs in vitro with said antigenic peptide(s) and/or immunogeniccompound(s) (Rizzo et al., Ex vivo loading of autologous dendritic cellswith tumor antigens. Methods Mol Biol. 2014; 1139:41-4; Rolinski andHus, Breaking immunotolerance of tumors: a new perspective for dendriticcell therapy. J Immunotoxicol. 2014 October; 11(4):311-8).

Preferred antigen-presenting cells according to the invention aredendritic cells (DCs). It can indeed be advantageous to combine at leastone antigenic peptide or immunogenic compound according to the inventionwith dendritic cells, as those are the most potent antigen-presentingcells and have been reported to be frequently functionally defective incancer patients. Dendritic cells can be easily obtained by the skilledperson in the art from either healthy compatible donors (i.e. thedendritic cells are HLA-related) or from the patient himself providedthat they are functional (i.e. the dendritic cells are autologous), forexample by direct isolation from the peripheral blood, or by derivationfrom peripheral blood cells such as CD14+ monocytes or CD34+hematopoietic precursors (Emens et al., 2008). Dendritic cells canindeed be distinguished from other cells of peripheral blood by theirsurface markers, such as S100, p55, CD83, and/or OX62, and may thus beisolated and purified based on said markers using cell culturestechniques well-known in the art.

According to a preferred embodiment, the pharmaceutical composition mayfurther comprise at least one anti-cancer therapeutic agent. Saidtherapeutic agent is thus preferably capable of preventing and/ortreating the same type of cancer than the one for which the antigenicpeptide according to the invention is used. Particularly preferredanti-cancer therapeutic agents according to the invention include,without limitation, antibodies, tumor cell lysates, chemotherapeuticagents, radiotherapeutic agents and combinations thereof. Mostpreferably, the anti-cancer therapeutic agent is selected fromantibodies, tumor cell lysates, chemotherapeutic agents,radiotherapeutic agents, immune checkpoint modulators and combinationsthereof.

Antibodies are particularly advantageous in cancer therapy as they caneither bind to specific antigens on cancer cell surfaces, therebydirecting the therapy to the tumor (i.e. these are referred astumor-targeting antibodies), or block immune checkpoints that aredysregulated in cancer (i.e. these are referred herein asimmunomodulatory antibodies). The purpose of the later type ofantibodies is to inhibit cancer immune resistance, which can notably beobserved against T cells that are specific for tumor antigens. Indeed,as well-known in the art, under normal physiological conditions, immunecheckpoints are crucial for the maintenance of self-tolerance (i.e.prevention of autoimmunity) and protect tissues from damage when theimmune system is responding to pathogenic infection. However, in cancer,immune-checkpoints expression can be dysregulated as an importantmechanism of immune resistance. Said resistance has notably beenobserved in melanoma, ovarian, lung, glioblastoma, breast, andpancreatic cancers with regard to the PD-L1 checkpoint (Konishi et al.,B7-H1 expression on non-small cell lung cancer cells and itsrelationship with tumor-infiltrating lymphocytes and their PD-1expression. Clin Cancer Res. 2004 Aug. 1; 10(15):5094-100; Ghebeh etal., The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed inbreast cancer patients with infiltrating ductal carcinoma: correlationwith important high-risk prognostic factors. Neoplasia. 2006 March;8(3):190-8; Hino et al., Tumor cell expression of programmed celldeath-1 ligand 1 is a prognostic factor for malignant melanoma. Cancer.2010 Apr. 1; 116(7):1757-66). Other examples of immune checkpointsinclude, without limitation, PD-L2, PD1, CD80, CD86, CTLA4, B7H3, B7H4,PVR, TIGIT, GAL9, LAG-3, GITR, CD137, TIM3, VISTA, VISTA-R (Pico deCoaña et al., Checkpoint blockade for cancer therapy: revitalizing asuppressed immune system. Trends Mol Med. 2015 August; 21(8):482-91;Pardoll D M. The blockade of immune checkpoints in cancer immunotherapy.Nat Rev Cancer. 2012 Mar. 22; 12(4):252-64).

Antibodies are usually employed for the above purposes either in theform of naked monoclonal antibodies (i.e. non-conjugated), or conjugatedto another molecule which can be toxic to cells or radioactive.

Examples of well-known monoclonal tumor-targeting antibodies used incancer immunotherapy include, without limitation, alemtuzumab (chroniclymphocytic leukemia), bevacizumab (colorectal cancer, glioblastomamultiforme, cervical cancer, lung cancer, renal cancer),brentuximab/vedotin (lymphomas), blinatumumab (acute lymphoblasticleukemia), catumaxomab (malignant ascites in EPCAM+ cancers), cetuximab(head and neck cancer, colorectal cancer), denosumab (breast, prostateand bone cancers), Gemtuzumab/ozogamicin (acute myeloid keulemia),ibritumomab/tiuxetan (non-Hodgkin lymphoma), panitumumab (colorectalcancer), pertuzumab (breast cancer), obinutuzumab (chronic lymphocyticleukemia), ofatumumab (chronic lymphocytic leukemia), opilimumab(melanoma), ramucirumab (gastric and gastro-oeasophageal cancers),rituximab (chronic lymphocytic leukemia and non-Hodgkin lymphoma),siltuximab (multicentric's Catsleman's disease), tositumomab(non-Hodgkin lymphoma), and trastuzumab (breast, gastric andgastro-oeasophageal cancers); while examples of immunomodulatoryantibodies include, without limitation, ipilimumab (melanoma) whichblocks the CTLA4-dependent immune checkpoint, nivolumab (melanoma, lungcancer) and prembrolizubmab (melanoma) which both block thePDCD1-dependent immune checkpoint, as well as MPDL3280A, MEDI4736,MEDI0680, and MSB0010718C which all block the PD-L1-dependent immunecheckpoint (Sharma and Allison, The future of immune checkpoint therapy.Science. 2015 Apr. 3; 348(6230):56-61).

Other antibodies for cancer immunotherapy have been described in Buqueet al., Trial Watch: Immunomodulatory monoclonal antibodies foroncological indications. Oncoimmunology. 2015 Mar. 2; 4(4):e1008814.eCollection 2015 April; Redman et al., Mechanisms of action oftherapeutic antibodies for cancer. Mol Immunol. 2015 October; 67(2 PtA):28-45; Simpson and Caballero, Monoclonal antibodies for the therapyof cancer MC Proc. 2014; 8(Suppl 4): 06 as well as on the antibodysociety website (list of therapeutic monoclonal antibodies approved orin review in the European Union or United States available on theweblink http://www.antibodysociety.org/news/approved_mabs.php).

Tumor cell lysates may also be combined with the antigenic peptide(s)according to the invention. Tumor cells are indeed capable of primingthe immune response, by presenting endogenous peptides-MHC complexes, aswell as via dendritic cells (DCs) of the host which can process andpresent the antigen delivered by said lysates. The range of antigensagainst which an immune response can be induced is thereby increased.Tumor cell lysates can be easily obtained by treating tumor cells with aheat shock and/or a chemical treatment, and can be autologous (i.e.isolated from the patient), or allogeneic (i.e. isolated from anothersubject).

Standard chemotherapeutic drugs and radiotherapeutic agents need not befurther described herein as they have been extensively described in theliterature, notably by Baskar et al. (Baskar et al., Cancer andradiation therapy: current advances and future directions. Int J MedSci. 2012; 9(3):193-9), Paci et al., (Paci et al., Review of therapeuticdrug monitoring of anticancer drugs part 1—cytotoxics. Eur J Cancer.2014 August; 50(12):2010-9) and Widmer et al. (Widmer et al., Review oftherapeutic drug monitoring of anticancer drugs part two—targetedtherapies. Eur J Cancer. 2014 August; 50(12):2020-36). A list of suchdrugs and agents is also available on the cancer.gov website(http://www.cancer.gov/about-cancer/treatment/drugs).

Preferably, the immune checkpoint modulator for combination with theantigenic peptide as defined herein is an activator or an inhibitor ofone or more immune checkpoint point molecule(s) selected from CD27,CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA,CD40, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, CEACAM1, GARP, PS,CSF1R, CD94/NKG2A, TDO, GITR, TNFR and/or FasR/DcR3; or an activator oran inhibitor of one or more ligands thereof.

More preferably, the immune checkpoint modulator is an activator of a(co-)stimulatory checkpoint molecule or an inhibitor of an inhibitorycheckpoint molecule or a combination thereof. Accordingly, the immunecheckpoint modulator is more preferably (i) an activator of CD27, CD28,CD40, CD122, CD137, OX40, GITR and/or ICOS or (ii) an inhibitor of A2AR,B7-H3, B7-H4, BTLA, CD40, CTLA-4, IDO, KIR, LAG3, PD-1, PDL-1, PD-L2,TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR and/orFasR/DcR3.

Even more preferably, the immune checkpoint modulator is an inhibitor ofan inhibitory checkpoint molecule (but preferably no inhibitor of astimulatory checkpoint molecule). Accordingly, the immune checkpointmodulator is even more preferably an inhibitor of A2AR, B7-H3, B7-H4,BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PDL-1, PD-L2, TIM-3, VISTA, CEACAM1,GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR and/or DcR3 or of a ligandthereof.

It is also preferred that the immune checkpoint modulator is anactivator of a stimulatory or costimulatory checkpoint molecule (butpreferably no activator of an inhibitory checkpoint molecule).Accordingly, the immune checkpoint modulator is more preferably anactivator of CD27, CD28, CD40, CD122, CD137, OX40, GITR and/or ICOS orof a ligand thereof.

It is even more preferred that the immune checkpoint modulator is amodulator of the CD40 pathway, of the IDO pathway, of the LAG3 pathway,of the CTLA-4 pathway and/or of the PD-1 pathway. In particular, theimmune checkpoint modulator is preferably a modulator of CD40, LAG3,CTLA-4, PD-L1, PD-L2, PD-1 and/or IDO, more preferably the immunecheckpoint modulator is an inhibitor of CTLA-4, PD-L1, PD-L2, PD-1,LAG3, and/or IDO or an activator of CD40, even more preferably theimmune checkpoint modulator is an inhibitor of CTLA-4, PD-L1, PD-1, LAG3and/or IDO, even more preferably the immune checkpoint modulator is aninhibitor of LAG3, CTLA-4 and/or PD-1, and most preferably the immunecheckpoint modulator is an inhibitor of CTLA-4 and/or PD-1.

Accordingly, the checkpoint modulator for combination with the antigenicpeptide may be selected from known modulators of the CTLA-4 pathway orthe PD-1 pathway. Preferably, the checkpoint modulator for combinationwith the antigenic peptide as defined herein may be selected from knownmodulators of the CTLA-4 pathway or the PD-1 pathway. Particularlypreferably, the immune checkpoint modulator is a PD-1 inhibitor.Preferred inhibitors of the CTLA-4 pathway and of the PD-1 pathwayinclude the monoclonal antibodies Yervoy® (Ipilimumab; Bristol MyersSquibb) and Tremelimumab (Pfizer/MedImmune) as well as Opdivo®(Nivolumab; Bristol Myers Squibb), Keytruda® (Pembrolizumab; Merck),Durvalumab (MedImmune/AstraZeneca), MEDI4736 (AstraZeneca; cf. WO2011/066389 A1), MPDL3280A (Roche/Genentech; cf. U.S. Pat. No. 8,217,149B2), Pidilizumab (CT-011; CureTech), MEDI0680 (AMP-514; AstraZeneca),MSB-0010718C (Merck), MIH1 (Affymetrix) and Lambrolizumab (e.g.disclosed as hPD109A and its humanized derivatives h409All, h409A16 andh409A17 in WO2008/156712; Hamid et al., 2013; N. Engl. J. Med. 369:134-144). More preferred checkpoint inhibitors include the CTLA-4inhibitors Yervoy® (Ipilimumab; Bristol Myers Squibb) and Tremelimumab(Pfizer/MedImmune) as well as the PD-1 inhibitors Opdivo® (Nivolumab;Bristol Myers Squibb), Keytruda® (Pembrolizumab; Merck), Pidilizumab(CT-011; CureTech), MEDI0680 (AMP-514; AstraZeneca), AMP-224 andLambrolizumab (e.g. disclosed as hPD109A and its humanized derivativesh409All, h409A16 and h409A17 in WO2008/156712; Hamid O. et al., 2013; N.Engl. J. Med. 369: 134-144.

It is also preferred that the immune checkpoint modulator forcombination with the antigenic peptide as defined herein is selectedfrom the group consisting of Pembrolizumab, Ipilimumab, Nivolumab,MPDL3280A, MEDI4736, Tremelimumab, Avelumab, PDR001, LAG525, INCB24360,Varlilumab, Urelumab, AMP-224 and CM-24.

It is within the skill of ordinary person in the art to select theappropriate immune anti-cancer therapeutic agent for the purposes of theinvention. For example, should one wish to prevent or treat melanoma, alysate from melanoma cells and/or the antibody opilimumab can preferablybe used, along with the corresponding antigenic peptide as described inTable 1A.

The anti-cancer therapeutic agent can also be administered inassociation with the composition of the invention, eithersimultaneously, separately, or sequentially. Should the composition andthe therapeutic agent be administered in a separate or sequentialmanner, those may be administered in distinct pharmaceutical forms.

Thus, in another aspect, the invention relates to a composition of theinvention and at least one anti-cancer therapeutic agent as describedabove, as a combined preparation for a simultaneous, separate, orsequential administration. In other terms, the invention proposes acombined use of the composition the invention and least one anti-cancertherapeutic agent as described above, for a simultaneous, separate, orsequential administration.

In a further aspect, the present invention also relates to akit-of-parts, preferably for use in the prevention and/or treatment ofcancer, the kit comprising at least one of:

-   -   an immunogenic compound according to the present invention,    -   an antigenic peptide according to the present invention,    -   a nanoparticle according to the present invention,    -   a cell according to the present invention,    -   a nucleic acid according to the present invention,    -   a host cell according to the present invention, or    -   an immunogenic composition according to the present invention.

In particular, the kit-of-parts of the invention may comprise more thanone of the above described components. For example, the kit-of-partsaccording to the present invention may comprise at least two differentimmunogenic compounds, at least two different antigenic peptides, atleast two different nanoparticles, at least two different cells, atleast two different nucleic acids, at least two different host cells,and/or at least two different immunogenic compositions. Preferably, suchdifferent components comprised by the kit-of-parts as described abovediffer in the antigenic peptides according to the present invention, forexample one component relating to a first antigenic peptide, and onecomponent relating to a second antigenic peptide (distinct from thefirst antigenic peptide).

For example, the kit may comprise two distinct immunogenic compoundsaccording to the present invention.

For example, the kit may comprise two distinct antigenic peptidesaccording to the present invention.

For example, the kit may comprise two distinct nanoparticles accordingto the present invention.

For example, the kit may comprise two distinct nucleic acids accordingto the present invention.

The various components of the kit-of-parts may be packaged in one ormore containers. The above components may be provided in a lyophilizedor dry form or dissolved in a suitable buffer. The kit may also compriseadditional reagents including, for instance, preservatives, growthmedia, and/or buffers for storage and/or reconstitution of theabove-referenced components, washing solutions, and the like. Inaddition, the kit-of-parts according to the present invention mayoptionally contain instructions of use.

Moreover, the present invention also provides a vaccination kit fortreating, preventing and/or stabilizing a cancer, comprising theimmunogenic composition as described herein or a vaccine as describedherein and instructions for use of said immunogenic composition or ofsaid vaccine in the prevention and/or treatment of a cancer.

Preferably, such a kit further comprises a package insert or instructionleaflet with directions to prevent or to treat a cancer by using theimmunogenic compound according to the present invention, the antigenicpeptide according to the present invention, the nanoparticle accordingto the present invention, the cell according to the present invention,the nucleic acid according to the present invention, the host cellaccording to the present invention, or the immunogenic compositionaccording to the present invention.

It is also preferred that, in addition to any of components as describedabove, the kit comprises an anti-cancer therapeutic agent as describedherein.

Medical Treatment and Uses

As stated above, the composition of the invention can be particularlyuseful for therapeutic purposes, notably for triggering a specificimmune response towards a particular tumor antigen/protein, so as toprevent or treat cancer in a patient in need thereof.

In a further aspect the present invention provides an immunogeniccompound according to the present invention, an antigenic peptideaccording to the present invention, a nanoparticle according toaccording to the present invention, a cell according to the presentinvention, a nucleic acid according to the present invention, a hostcell according to the present invention, or an immunogenic compositionaccording to the present invention, for use in the prevention and/or inthe treatment of a cancer. Preferably said cancer relates to the(reference) antigen of the antigenic peptide as described above.

Accordingly, the present invention provides a method for preventingand/or treating a cancer or initiating, enhancing or prolonging ananti-tumor-response in a subject in need thereof comprisingadministering to the subject

-   -   the immunogenic compound according to the present invention,    -   the antigenic peptide according to the present invention,    -   the nanoparticle according to the present invention,    -   the cell according to the present invention,    -   the nucleic acid according to the present invention,    -   the host cell according to the present invention,    -   the immunogenic composition according to the present invention,        or    -   the combination according to the present invention as described        herein.

Moreover, the present invention provides a method for eliciting orimproving, in a subject, an immune response against one or multipleepitopes that is dependent on CD8⁺ cytotoxic T cells, wherein saidmethod comprises administering to said subject any one of:

-   -   the immunogenic compound according to the present invention,    -   the antigenic peptide according to the present invention,    -   the nanoparticle according to the present invention,    -   the cell according to the present invention,    -   the nucleic acid according to the present invention,    -   the host cell according to the present invention,    -   the immunogenic composition according to the present invention,        or    -   the combination according to the present invention as described        herein.

An immune response that is dependent on CD8⁺ response can be determinedby evaluating an inflammatory response, a pro-inflammatory cytokineresponse, including an increase in the expression of one or more ofIFN-γ, TNF-α and IL-2 mRNA or protein relative to the level beforeadministration of the compounds of the invention. It can also bemeasured by an increase in the frequency or absolute number ofantigen-specific T cells after administration of the compounds of theinvention, measured by HLA-peptide multimer staining, ELISPOT assays,and delayed type hypersensitivity tests. It can also be indirectlymeasured by an increase in antigen-specific serum antibodies that aredependent on antigen-specific T helper cells.

The present invention also provides a method for eliciting or improving,in a subject, an immune response against one or multiple antigens orantigenic epitopes that is restricted by multiple MHC class I molecules,wherein said method comprises administering to said subject any one of

-   -   the immunogenic compound according to the present invention,    -   the antigenic peptide according to the present invention,    -   the nanoparticle according to the present invention,    -   the cell according to the present invention,    -   the nucleic acid according to the present invention,    -   the host cell according to the present invention,    -   the immunogenic composition according to the present invention,        or    -   the combination according to the present invention as described        herein.

A method for eliciting or improving, in a subject, an immune responseagainst multiple epitopes as described herein, that is restricted bymultiple MHC class I molecules can be determined by evaluating acytokine response, including an increase in the expression of one ormore of IFN-γ, TNF-α and IL-2 mRNA or protein relative to the levelbefore administration of the compounds of the invention, after in vitrostimulation of T cells with individual peptides binding to discrete MHCclass I molecules on antigen presenting cells. Restriction to MHC classI molecules can also be validated by using antigen presenting cellsexpressing MHC class I molecules, or by using MHC class I blockingantibodies. It can also be measured by an increase in the frequency orabsolute number of antigen-specific T cells after administration of thecompounds of the invention, measured by HLA-peptide multimer staining,using multimers assembled with MHC class I molecules.

Thus, in another aspect, the invention relates to a composition asdefined above, for use as a medicament. Moreover,

-   -   the immunogenic compound according to the present invention,    -   the antigenic peptide according to the present invention,    -   the nanoparticle according to the present invention,    -   the cell according to the present invention,    -   the nucleic acid according to the present invention,    -   the host cell according to the present invention,    -   the immunogenic composition according to the present invention,        or    -   the combination according to the present invention as described        herein may be used as a medicament.

The invention relates more particularly to a composition as definedabove, for use as a vaccine for immunotherapy. Moreover,

-   -   the immunogenic compound according to the present invention,    -   the antigenic peptide according to the present invention,    -   the nanoparticle according to the present invention,    -   the cell according to the present invention,    -   the nucleic acid according to the present invention,    -   the host cell according to the present invention,    -   the immunogenic composition according to the present invention,        or    -   the combination according to the present invention as described        herein may be used as vaccine, in particular for (cancer)        immunotherapy.

As used in the context of the present invention, the term “vaccine”refers to a biological preparation that provides innate and/or adaptiveimmunity, typically to a particular disease, preferably cancer. Thus, avaccine supports in particular an innate and/or an adaptive immuneresponse of the immune system of a subject to be treated. For example,the antigenic peptide according to the present invention typically leadsto or supports an adaptive immune response in the patient to be treated.

In the context of the present invention, the vaccine (composition) caninduce a specific immune response against a tumor antigen, and is thuspreferably used to prevent or treat cancer. It can also be referredherein as a cancer vaccine.

Accordingly, in a preferred embodiment, the invention relates to acomposition as defined above, for use in the prevention and/or treatmentof cancer in a subject in need thereof. More precisely, the inventionrelates to the use of the composition of the invention for manufacturinga medicament to prevent or treat cancer in a subject in need thereof.

In other words, the invention relates to a method for preventing ortreating cancer in a subject in need thereof, comprising administeringan effective amount of the composition of the invention, to saidsubject.

Methods of administration of a medicament are well-known to the skilledperson in the art. With regard to the composition of the invention, itcan be directly administered into the subject, into the affected organ(i.e. local administration) or systemically (i.e. enteral or parenteraladministration), or even applied ex vivo to cells derived from thesubject or a human cell line which are subsequently administered to thesubject, or even used in vitro to select a subpopulation of immune cellsderived from the subject, which are then re-administered to the saidsubject. Enteral administrations as used herein includes oral and rectaladministrations, as well as administrations via gastric feeding tubes,duodenal feeding tubes or gastrostomy, while parenteral administrationsincludes, among others, subcutaneous, intravenous, intramuscular,intra-arterial, intradermal, intraosseous, intracerebral, andintrathecal injections. The administration method will often depend uponthe antigenic peptide(s) and/or immunogenic compound(s) present in thecomposition, and the type of cancer to be treated and other activeagents that may be contained in said composition. For example, theadministration is preferably an intramuscular or an intradermalinjection if the immunogenic compound is a nucleic acid as definedabove, the oral/nasal administration being particularly preferred ifsaid nucleic acid is cloned into a viral vector. Alternatively, theadministration is preferably an intramuscular, an intradermal or an oraladministration if the antigenic peptide and/or immunogenic compound is a(poly)peptide as defined above or if it is loaded in/on a nanoparticleas described herein. Yet, still alternatively, the administration ispreferably an oral administration if the antigenic peptide and/orimmunogenic compound is delivered in the form of a gut bacterium asdefined above, notably if the gut bacterium is in the form ofprobiotics.

The antigenic peptides and/or immunogenic compounds according to theinvention can further be encapsulated so as to facilitate theiradministration to the subject in need thereof. For example, those may beencapsulated into peptide nanocarriers (preferable if the immunogeniccompound is a nucleic acid or a (poly)peptide), into virosomes(preferable if the immunogenic compound is a nucleic acid or a(poly)peptide), or into lipid-based carrier systems such asliposome-polycation-DNA complex (preferable if the immunogen is anucleic acid or a (poly)peptide) (Trovato M, De Berardinis P. Novelantigen delivery systems. World J Virol. 2015 Aug. 12; 4(3):156-68;Saade F, Petrovsky N. Technologies for enhanced efficacy of DNAvaccines. Expert Rev Vaccines. 2012 February; 11(2):189-209; Li et al.,Peptide Vaccine: Progress and Challenges. Vaccines (Basel). 2014 Jul. 2;2(3):515-36).

The composition may also be administered more than once so as to achievethe desired effect. In a preferred embodiment, said composition isadministered repeatedly, at least twice, and preferably more than twice.This can be done over an extended period of time, such as weekly, everyother week, monthly, yearly, or even several years after the firstadministration to ensure that the subject is properly immunized.

According to one embodiment, an antigenic peptide or an immunogeniccompound according to the invention may be used for the preparation of acomposition and/or of an immunogenic composition for preventing ortreating cancer in a subject in need thereof.

Combination Therapy

The administration of the antigenic peptide according to the presentinvention, the immunogenic compound according to the present invention,the nanoparticle according to the present invention, the cell accordingto the present invention, the nucleic acid according to the presentinvention, the host cell according to the present invention, and theimmunogenic composition according to the present invention, inparticular in the methods and uses according to the invention, can becarried out alone or in combination with a co-agent useful for treatingand/or preventing cancer, such as an anti-cancer therapeutic agent.

Said therapeutic agent is thus preferably capable of preventing and/ortreating the same type of cancer as the one for which the antigenicpeptide according to the invention is used. Particularly preferredanti-cancer therapeutic agents according to the invention include,without limitation, antibodies, tumor cell lysates, chemotherapeuticagents, radiotherapeutic agents, immune checkpoint modulators andcombinations thereof.

Antibodies are particularly advantageous in cancer therapy as they caneither bind to specific antigens on cancer cell surfaces, therebydirecting the therapy to the tumor (i.e. these are referred astumor-targeting antibodies), or block immune checkpoints that aredysregulated in cancer (i.e. these are referred herein asimmunomodulatory antibodies). The purpose of the later type ofantibodies is to inhibit cancer immune resistance, which can notably beobserved against T cells that are specific for tumour antigens. Indeed,as well-known in the art, under normal physiological conditions, immunecheckpoints are crucial for the maintenance of self-tolerance (i.e.prevention of autoimmunity) and protect tissues from damage when theimmune system is responding to pathogenic infection. However, in cancer,immune-checkpoints expression can be dysregulated as an importantmechanism of immune resistance. Said resistance has notably beenobserved in melanoma, ovarian, lung, glioblastoma, breast, andpancreatic cancers with regard to the PD-L1 checkpoint (Konishi et al.,B7-H1 expression on non-small cell lung cancer cells and itsrelationship with tumor-infiltrating lymphocytes and their PD-1expression. Clin Cancer Res. 2004 Aug. 1; 10(15):5094-100; Ghebeh etal., The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed inbreast cancer patients with infiltrating ductal carcinoma: correlationwith important high-risk prognostic factors. Neoplasia. 2006 March;8(3):190-8; Hino et al., Tumor cell expression of programmed celldeath-1 ligand 1 is a prognostic factor for malignant melanoma. Cancer.2010 Apr. 1; 116(7):1757-66). Other examples of immune checkpointsinclude, without limitation, PD-L2, PD1, CD80, CD86, CTLA4, B7H3, B7H4,PVR, TIGIT, GAL9, LAG-3, GITR, CD137, TIM3, VISTA, VISTA-R (Pico deCoaña et al., Checkpoint blockade for cancer therapy: revitalizing asuppressed immune system. Trends Mol Med. 2015 August; 21(8):482-91;Pardoll D M1. The blockade of immune checkpoints in cancerimmunotherapy. Nat Rev Cancer. 2012 Mar. 22; 12(4):252-64).

Antibodies are usually employed for the above purposes either in theform of naked monoclonal antibodies (i.e. non-conjugated), or conjugatedto another molecule which can be toxic to cells or radioactive.

Examples of well-known monoclonal tumor-targeting antibodies used incancer immunotherapy include, without limitation, alemtuzumab (chroniclymphocytic leukemia), bevacizumab (colorectal cancer, glioblastomamultiforme, cervical cancer, lung cancer, renal cancer),brentuximab/vedotin (lymphomas), blinatumumab (acute lymphoblasticleukemia), catumaxomab (malignant ascites in EPCAM+ cancers), cetuximab(head and neck cancer, colorectal cancer), denosumab (breast, prostateand bone cancers), Gemtuzumab/ozogamicin (acute myeloid keulemia),ibritumomab/tiuxetan (non-Hodgkin lymphoma), panitumumab (colorectalcancer), pertuzumab (breast cancer), obinutuzumab (chronic lymphocyticleukemia), ofatumumab (chronic lymphocytic leukemia), opilimumab(melanoma), ramucirumab (gastric and gastro-oeasophageal cancers),ntuximab (chronic lymphocytic leukemia and non-Hodgkin lymphoma),siltuximab (multicentric's Catsleman's disease), tositumomab(non-Hodgkin lymphoma), and trastuzumab (breast, gastric andgastro-oeasophageal cancers); while examples of immunomodulatoryantibodies include, without limitation, ipilimumab (melanoma) whichblocks the CTLA4-dependent immune checkpoint, nivolumab (melanoma, lungcancer) and prembrolizubmab (melanoma) which both block thePDCD1-dependent immune checkpoint, as well as MPDL3280A, MEDI4736,MEDI0680, and MSB0010718C which all block the PD-L1-dependent immunecheckpoint (Sharma and Allison, The future of immune checkpoint therapy.Science. 2015 Apr. 3; 348(6230):56-61).

Other antibodies for cancer immunotherapy have been described in Buqueet al. (Buque et al., Trial Watch: Immunomodulatory monoclonalantibodies for oncological indications. Oncoimmunology. 2015 Mar. 2;4(4):e1008814. eCollection 2015 April), Redman et al. (Redman et al.,Mechanisms of action of therapeutic antibodies for cancer. Mol Immunol.2015 October; 67(2 Pt A):28-45), and in Simpson and Caballero,Monoclonal antibodies for the therapy of cancer MC Proc. 2014; 8(Suppl4): 06 as well as on the antibody society website (list of therapeuticmonoclonal antibodies approved or in review in the European Union orUnited States available on the weblinkhttp://www.antibodysociety.org/news/approved_mabs.php).

Tumor cell lysates may also be combined with the antigenic peptide(s)according to the invention. Tumor cells are indeed capable of primingthe immune response, by presenting endogenous peptides-MHC complexes, aswell as via dendritic cells (DCs) of the host which can process andpresent the antigen delivered by said lysates. The range of antigensagainst which an immune response can be induced is thereby increased.Tumor cell lysates can be easily obtained by treating tumor cells with aheat shock and/or a chemical treatment, and can be autologous (i.e.isolated from the patient), or allogeneic (i.e. isolated from anothersubject).

Standard chemotherapeutic drugs and radiotherapeutic agents need not befurther described herein as they have been extensively described in theliterature, notably by Baskar et al. (Baskar et al., Cancer andradiation therapy: current advances and future directions. Int J MedSci. 2012; 9(3):193-9), Paci et al. (Paci et al., Review of therapeuticdrug monitoring of anticancer drugs part 1—cytotoxics. Eur J Cancer.2014 August; 50(12):2010-9) and Widmer et al. (Widmer et al., Review oftherapeutic drug monitoring of anticancer drugs part two—targetedtherapies. Eur J Cancer. 2014 August; 50(12):2020-36). A list of suchdrugs and agents is also available on the cancer.gov website(http://www.cancer.gov/about-cancer/treatment/drugs).

Preferably, the immune checkpoint modulator for combination with theantigenic peptide as defined herein is an activator or an inhibitor ofone or more immune checkpoint point molecule(s) selected from CD27,CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA,CD40, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, CEACAM1, GARP, PS,CSF1R, CD94/NKG2A, TDO, GITR, TNFR and/or FasR/DcR3; or an activator oran inhibitor of one or more ligands thereof.

More preferably, the immune checkpoint modulator is an activator of a(co-)stimulatory checkpoint molecule or an inhibitor of an inhibitorycheckpoint molecule or a combination thereof. Accordingly, the immunecheckpoint modulator is more preferably (i) an activator of CD27, CD28,CD40, CD122, CD137, OX40, GITR and/or ICOS or (ii) an inhibitor of A2AR,B7-H3, B7-H4, BTLA, CD40, CTLA-4, IDO, KIR, LAG3, PD-1, PDL-1, PD-L2,TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR and/orFasR/DcR3.

Even more preferably, the immune checkpoint modulator is an inhibitor ofan inhibitory checkpoint molecule (but preferably no inhibitor of astimulatory checkpoint molecule). Accordingly, the immune checkpointmodulator is even more preferably an inhibitor of A2AR, B7-H3, B7-H4,BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PDL-1, PD-L2, TIM-3, VISTA, CEACAM1,GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR and/or DcR3 or of a ligandthereof.

It is also preferred that the immune checkpoint modulator is anactivator of a stimulatory or costimulatory checkpoint molecule (butpreferably no activator of an inhibitory checkpoint molecule).Accordingly, the immune checkpoint modulator is more preferably anactivator of CD27, CD28, CD40, CD122, CD137, OX40, GITR and/or ICOS orof a ligand thereof.

It is even more preferred that the immune checkpoint modulator is amodulator of the CD40 pathway, of the IDO pathway, of the LAG3 pathway,of the CTLA-4 pathway and/or of the PD-1 pathway. In particular, theimmune checkpoint modulator is preferably a modulator of CD40, LAG3,CTLA-4, PD-L1, PD-L2, PD-1 and/or IDO, more preferably the immunecheckpoint modulator is an inhibitor of CTLA-4, PD-L1, PD-L2, PD-1,LAG3, and/or IDO or an activator of CD40, even more preferably theimmune checkpoint modulator is an inhibitor of CTLA-4, PD-L1, PD-1, LAG3and/or IDO, even more preferably the immune checkpoint modulator is aninhibitor of LAG3, CTLA-4 and/or PD-1, and most preferably the immunecheckpoint modulator is an inhibitor of CTLA-4 and/or PD-1.

Accordingly, the checkpoint modulator for combination with the antigenicpeptide may be selected from known modulators of the CTLA-4 pathway orthe PD-1 pathway. Preferably, the checkpoint modulator for combinationwith the antigenic peptide as defined herein may be selected from knownmodulators of the CTLA-4 pathway or the PD-1 pathway. Particularlypreferably, the immune checkpoint modulator is a PD-1 inhibitor.Preferred inhibitors of the CTLA-4 pathway and of the PD-1 pathwayinclude the monoclonal antibodies Yervoy® (Ipilimumab; Bristol MyersSquibb) and Tremelimumab (Pfizer/MedImmune) as well as Opdivo®(Nivolumab; Bristol Myers Squibb), Keytruda® (Pembrolizumab; Merck),Durvalumab (MedImmune/AstraZeneca), MEDI4736 (AstraZeneca; cf. WO2011/066389 A1), MPDL3280A (Roche/Genentech; cf. U.S. Pat. No. 8,217,149B2), Pidilizumab (CT-011; CureTech), MEDI0680 (AMP-514; AstraZeneca),MSB-0010718C (Merck), MIH1 (Affymetrix) and Lambrolizumab (e.g.disclosed as hPD109A and its humanized derivatives h409All, h409A16 andh409A17 in WO2008/156712; Hamid et al., 2013; N. Engl. J. Med. 369:134-144). More preferred checkpoint inhibitors include the CTLA-4inhibitors Yervoy® (Ipilimumab; Bristol Myers Squibb) and Tremelimumab(Pfizer/MedImmune) as well as the PD-1 inhibitors Opdivo® (Nivolumab;Bristol Myers Squibb), Keytruda® (Pembrolizumab; Merck), Pidilizumab(CT-011; CureTech), MEDI0680 (AMP-514; AstraZeneca), AMP-224 andLambrolizumab (e.g. disclosed as hPD109A and its humanized derivativesh409All, h409A16 and h409A17 in WO2008/156712; Hamid O. et al., 2013; N.Engl. J. Med. 369: 134-144.

It is also preferred that the immune checkpoint modulator forcombination with the antigenic peptide as defined herein is selectedfrom the group consisting of Pembrolizumab, Ipilimumab, Nivolumab,MPDL3280A, MEDI4736, Tremelimumab, Avelumab, PDR001, LAG525, INCB24360,Varlilumab, Urelumab, AMP-224 and CM-24.

It is within the skill of ordinary person in the art to select theappropriate immune anti-cancer therapeutic agent for the purposes of theinvention. For example, should one wish to prevent or treat melanoma, alysate from melanoma cells and/or the antibody opilimumab can preferablybe used, along with the corresponding antigenic peptide according to thepresent invention as described herein.

The anti-cancer therapeutic agent can also be administered inassociation with the antigenic peptide according to the presentinvention, the immunogenic compound according to the present invention,the nanoparticle according to the present invention, the cell accordingto the present invention, the nucleic acid according to the presentinvention, the host cell according to the present invention, or theimmunogenic composition according to the present invention, either atabout the same time or consecutively as described herein and in the sameor distinct pharmaceutical forms.

Thus, in another aspect, the invention relates to a composition of theinvention and at least one anti-cancer therapeutic agent as describedabove, as a combined preparation for a simultaneous, separate, orsequential administration. In other terms, the invention proposes acombined use of the composition the invention and least one anti-cancertherapeutic agent as described above, for a simultaneous, separate, orsequential administration.

Moreover, the present invention also provides the combination of (atleast) two distinct antigenic peptides according to the presentinvention as described herein. In this context, the (at least) twodistinct antigenic peptides may be in any form, e.g., “naked”, comprisedin immunogenic compounds, nanoparticles, (immunogenic) compositions orcells loaded therewith, or encoded by nucleic acids (e.g., vectors).Accordingly, the (at least) two distinct antigenic peptides may becomprised in (at least) two distinct components (to be combined).Accordingly, the two distinct components of the combination according tothe present invention refer in particular to distinct antigenic peptidesaccording to the present invention (which are comprised by theimmunogenic compounds, the nanoparticles, encoded by the nucleic acids,etc.). Such two distinct components, in particular the two distinctantigenic peptides according to the invention (comprised in the twodistinct components), relate preferably to the same type of cancer, forexample to the same or distinct antigens associated with this cancerand/or to the same or distinct (reference) epitopes within an antigenassociated with this cancer. More preferably, the two distinctcomponents, in particular the two distinct antigenic peptides accordingto the invention (comprised in the two distinct components), relate tothe same tumor (associated or specific) antigen. The two distinctcomponents, in particular the two distinct antigenic peptides accordingto the invention (comprised in the two distinct components), may alsorelate to the same or distinct (reference) tumor (associated orspecific) antigen(s).

Moreover, the antigenic peptide according to the present invention mayalso be combined with the corresponding (human) tumor antigen epitope(as described above regarding the peptide “families”). Thereby,selection of T-cell clones, which are very efficient against the tumor,is obtained/supported. In particular, the antigenic peptide according tothe present invention and the corresponding (human) tumor antigenepitope may be co-administered. Such co-administration may be at aboutthe same time (simultaneously) or consecutively, whereby in consecutiveadministration it is preferred that the antigenic peptide according tothe present invention is administered first and the corresponding(human) tumor antigen epitope is administered thereafter. In particular,the antigenic peptide according to the present invention may beadministered first, and the corresponding (human) tumor antigen epitopemay be used as (re)boost. For example, the antigenic peptide accordingto SEQ ID NO: 47 may be combined with the reference peptide according toSEQ ID NO: 120. In another example, the antigenic peptide according toSEQ ID NO: 51, 52, 55, or 56 may be combined with the reference peptideaccording to SEQ ID NO: 122. In another example, the antigenic peptideaccording to SEQ ID NO: 77 may be combined with the reference peptideaccording to SEQ ID NO: 128. In another example, the antigenic peptideaccording to SEQ ID NO: 93 may be combined with the reference peptideaccording to SEQ ID NO: 136. In another example, the antigenic peptideaccording to SEQ ID NO: 28 may be combined with the reference peptideaccording to SEQ ID NO: 115. In another example, the antigenic peptideaccording to SEQ ID NO: 101 or 102 may be combined with the referencepeptide according to SEQ ID NO: 141. In another example, the antigenicpeptide according to SEQ ID NO: 26 may be combined with the referencepeptide according to SEQ ID NO: 113.

Both peptides, which are to be combined, such as (a) the antigenicpeptide according to the present invention and the corresponding (human)tumor antigen epitope or (b) two distinct antigenic peptides accordingto the present invention, may be administered

-   -   in the same immunogenic compound according to the present        invention or in distinct immunogenic compounds according to the        present invention,    -   (loaded) in the same nanoparticle according to the present        invention or in distinct nanoparticles according to the present        invention,    -   (loaded) in the same cell according to the present invention or        in distinct cells according to the present invention,    -   (encoded by) the same nucleic acid according to the present        invention or by distinct nucleic acids according to the present        invention,    -   (expressed by) the same host cell according to the present        invention or by distinct host cells according to the present        invention, or    -   (comprised) in the same immunogenic composition according to the        present invention or in distinct immunogenic composition        according to the present invention.

For example, the present invention provides a combination of

-   -   (i) an immunogenic compound according to the present invention        comprising a first antigenic peptide according to the present        invention, and    -   (ii) an immunogenic compound according to the present invention        comprising a second antigenic peptide according to the present        invention for use in the prevention and/or treatment of a        cancer.

For example, the present invention provides a combination of

-   -   (i) a first antigenic peptide according to the present        invention, and    -   (ii) a second antigenic peptide according to the present        invention for use in the prevention and/or treatment of a        cancer.

For example, the present invention provides a combination of

(i) a nanoparticle according to the present invention comprising a firstantigenic peptide according to the present invention, and

(ii) a nanoparticle according to the present invention comprising asecond antigenic peptide according to the present invention

for use in the prevention and/or treatment of a cancer.

For example, the present invention provides a combination of

(i) a nucleic acid according to the present invention comprising apolynucleotide encoding a first antigenic peptide according to thepresent invention and

(ii) a nucleic acid according to the present invention comprising apolynucleotide encoding a first antigenic peptide according to thepresent invention

for use in the prevention and/or treatment of a cancer.

Preferably, both peptides, which are to be combined, such as (a) theantigenic peptide according to the present invention and thecorresponding (human) tumor antigen epitope or (b) two distinctantigenic peptides according to the present invention, in particularcomponents (i) and (ii), are administered at about the same time. Inmore general, it is preferred that the first (antigenic) peptide in anyformulation (e.g., in the form of the immunogenic compound according tothe present invention, the nanoparticle according to the presentinvention, the cell according to the present invention, the nucleic acidaccording to the present invention, the host cell according to thepresent invention, or the immunogenic composition according to thepresent invention; referred to herein as “the first (antigenic) peptidecomponent”) is administered at about the same time as the second(antigenic) peptide in any formulation (e.g., in the form of theimmunogenic compound according to the present invention, thenanoparticle according to the present invention, the cell according tothe present invention, the nucleic acid according to the presentinvention, the host cell according to the present invention, or theimmunogenic composition according to the present invention; referred toherein as “the second (antigenic) peptide component”), wherein both(antigenic) peptides are preferably administered in the same form (i.e.,in the same type of formulation, e.g., both as nanoparticles, both asimmunogenic compositions, etc.).

“At about the same time”, as used herein, means in particularsimultaneous administration or that directly after administration of (i)the first (antigenic) peptide component, (ii) the second (antigenic)peptide component is administered or directly after administration of(ii) the second (antigenic) peptide component (i) the first (antigenic)peptide component is administered. The skilled person understands that“directly after” includes the time necessary to prepare the secondadministration—in particular the time necessary for exposing anddisinfecting the location for the second administration as well asappropriate preparation of the “administration device” (e.g., syringe,pump, etc.). Simultaneous administration also includes if the periods ofadministration of (i) the first (antigenic) peptide component and of(ii) the second (antigenic) peptide component overlap or if, forexample, one component is administered over a longer period of time,such as 30 min, 1 h, 2 h or even more, e.g. by infusion, and the othercomponent is administered at some time during such a long period.Administration of (i) the first (antigenic) peptide component and of(ii) the second (antigenic) peptide component at about the same time isin particular preferred if different routes of administration and/ordifferent administration sites are used.

It is also preferred that both peptides, which are to be combined, suchas (a) the antigenic peptide according to the present invention and thecorresponding (human) tumor antigen epitope or (b) two distinctantigenic peptides according to the present invention, in particularcomponents (i) and (ii), are administered consecutively. In moregeneral, it is preferred that the first (antigenic) peptide in anyformulation (e.g., in the form of the immunogenic compound according tothe present invention, the nanoparticle according to the presentinvention, the cell according to the present invention, the nucleic acidaccording to the present invention, the host cell according to thepresent invention, or the immunogenic composition according to thepresent invention; referred to herein as “the first (antigenic) peptidecomponent”) and the second (antigenic) peptide in any formulation (e.g.,in the form of the immunogenic compound according to the presentinvention, the nanoparticle according to the present invention, the cellaccording to the present invention, the nucleic acid according to thepresent invention, the host cell according to the present invention, orthe immunogenic composition according to the present invention; referredto herein as “the second (antigenic) peptide component”) areadministered consecutively, wherein both (antigenic) peptides arepreferably administered in the same form (i.e., in the same type offormulation, e.g., both as nanoparticles, both as immunogeniccompositions, etc.).

This means that (i) the first (antigenic) peptide component isadministered before or after (ii) the second (antigenic) peptidecomponent. In consecutive administration, the time betweenadministration of the first component and administration of the secondcomponent is preferably no more than one week, more preferably no morethan 3 days, even more preferably no more than 2 days and mostpreferably no more than 24 h. It is particularly preferred that (i) thefirst (antigenic) peptide component and (ii) the second (antigenic)peptide component are administered at the same day with the time betweenadministration of the first component (the first or the second(antigenic) peptide) and administration of the second component (theother of the first or the second (antigenic) peptide) being preferablyno more than 6 hours, more preferably no more than 3 hours, even morepreferably no more than 2 hours and most preferably no more than 1 h.

Preferably, (i) the first (antigenic) peptide component and (ii) thesecond (antigenic) peptide component are administered via the same routeof administration. In more general, it is preferred that the first(antigenic) peptide in any formulation (e.g., in the form of theimmunogenic compound according to the present invention, thenanoparticle according to the present invention, the cell according tothe present invention, the nucleic acid according to the presentinvention, the host cell according to the present invention, or theimmunogenic composition according to the present invention; referred toherein as “the first (antigenic) peptide component”) and the second(antigenic) peptide in any formulation (e.g., in the form of theimmunogenic compound according to the present invention, thenanoparticle according to the present invention, the cell according tothe present invention, the nucleic acid according to the presentinvention, the host cell according to the present invention, or theimmunogenic composition according to the present invention; referred toherein as “the second (antigenic) peptide component”) are administeredvia the same route of administration, wherein both (antigenic) peptidesare preferably administered in the same form (i.e., in the same type offormulation, e.g., both as nanoparticles, both as immunogeniccompositions, etc.).

It is also preferred that components (i) and (ii) are administered viadistinct routes of administration. In more general, it is preferred thatthe first (antigenic) peptide in any formulation (e.g., in the form ofthe immunogenic compound according to the present invention, thenanoparticle according to the present invention, the cell according tothe present invention, the nucleic acid according to the presentinvention, the host cell according to the present invention, or theimmunogenic composition according to the present invention; referred toherein as “the first (antigenic) peptide component”) and the second(antigenic) peptide component in any formulation (e.g., in the form ofthe immunogenic compound according to the present invention, thenanoparticle according to the present invention, the cell according tothe present invention, the nucleic acid according to the presentinvention, the host cell according to the present invention, or theimmunogenic composition according to the present invention; referred toherein as “the second (antigenic) peptide component”) are administeredvia distinct routes of administration, wherein both (antigenic) peptidesare preferably administered in the same form (i.e., in the same type offormulation, e.g., both as nanoparticles, both as immunogeniccompositions, etc.).

Preferably, components (i) and (ii) are comprised in the samecomposition. In more general, it is preferred that the first (antigenic)peptide in any formulation (e.g., in the form of the immunogeniccompound according to the present invention, the nanoparticle accordingto the present invention, the cell according to the present invention,the nucleic acid according to the present invention, or the host cellaccording to the present invention; referred to herein as “the first(antigenic) peptide component”) and the second (antigenic) peptide inany formulation (e.g., in the form of the immunogenic compound accordingto the present invention, the nanoparticle according to the presentinvention, the cell according to the present invention, the nucleic acidaccording to the present invention, or the host cell according to thepresent invention; referred to herein as “the second (antigenic) peptidecomponent”) are comprised in the same composition, wherein both(antigenic) peptides are preferably administered in the same form (i.e.,in the same type of formulation, e.g., both as nanoparticles, etc.).

It is also preferred that components (i) and (ii) are comprised indistinct compositions. In more general, it is preferred that the first(antigenic) peptide in any formulation (e.g., in the form of theimmunogenic compound according to the present invention, thenanoparticle according to the present invention, the cell according tothe present invention, the nucleic acid according to the presentinvention, or the host cell according to the present invention; referredto herein as “the first (antigenic) peptide component”) and the second(antigenic) peptide in any formulation (e.g., in the form of theimmunogenic compound according to the present invention, thenanoparticle according to the present invention, the cell according tothe present invention, the nucleic acid according to the presentinvention, or the host cell according to the present invention; referredto herein as “the second (antigenic) peptide component”) are comprisedin distinct compositions, wherein both (antigenic) peptides arepreferably administered in the same form (i.e., in the same type offormulation, e.g., both as nanoparticles, etc.).

EXAMPLES

Examples 1 and 2 are both linked to the general protocol described inFIG. 1.

Example 1: Identification of a Candidate Antigenic Peptide HavingSuperior Affinity to the HLA-A*0201 Allele

This Example provides evidence that the antigenic peptide of sequenceSEQ ID NO:71 («FLPFGFILV» also referred herein as IL13RA2-B) has highaffinity to the HLA-A*0201 allele, whereas the corresponding referencehuman peptide derived from IL13RA2 («WLPFGFILI», SEQ ID NO:123, alsoreferred herein as IL13RA2-H) has low affinity.

A. Materials and Methods

A1. Measuring the Affinity of the Peptide to T2 Cell Line.

The experimental protocol is similar to the one that was validated forpeptides presented by the HLA-A*0201 (Tourdot et al., A general strategyto enhance immunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification of cryptic tumor epitopes. Eur JImmunol. 2000 December; 30(12):3411-21). Affinity measurement of thepeptides is achieved with the human tumoral cell T2 which expresses theHLA-A*0201 molecule, but which is TAP1/2 negative and incapable ofpresenting endogenous peptides.

T2 cells (2·10⁵ cells per well) are incubated with decreasingconcentrations of peptides from 100 μM to 0.1 μM in a AIMV mediumsupplemented with 100 ng/μl of human β2m at 37° C. for 16 hours. Cellsare then washed two times and marked with the anti-HLA-A2 antibodycoupled to PE (clone BB7.2, BD Pharmagen).

The analysis is achieved by FACS (Guava Easy Cyte).

For each peptide concentration, the geometric mean of the labellingassociated with the peptide of interest is subtracted from backgroundnoise and reported as a percentage of the geometric mean of theHLA-A*0202 labelling obtained for the reference peptide HIV pol 589-597at a concentration of 100 μM. The relative affinity is then determinedas follows:relative affinity=concentration of each peptide inducing 20% ofexpression of HLA-A*0201/concentration of the reference peptide inducing20% of expression of HLA-A*0201.

A2. Solubilisation of Peptides

Each peptide is solubilized by taking into account the amino acidcomposition. For peptides which do not include any Cystein, Methionin,or Tryptophane, the addition of DMSO is possible to up to 10% of thetotal volume. Other peptides are resuspended in water or PBS pH7.4.

B. Results

For T2 ATCC Cells: Mean fluorescence intensity for variable peptidicconcentrations: Regarding the couple IL13RA2 peptides (IL13RA2-H andIL13RA2-B), it appears that the human peptide does not bind to theHLA-A*0201 contrarily to the candidate peptide IL13RA2-B, which bindsstrongly to HLA-A*0201: 112.03 vs 18.64 at 100 μM; 40.77 vs 11.61 at 10μM; 12.18 vs 9.41 at 1 μM; 9.9 vs 7.46 at 0.1 μM.

Also, IL13RA2-B at 4.4 μM induces 20% of expression of the HLA-A*0201(vs 100 μM for IL13RA2-H).

Similar results were obtained from a second distinct T2 cell clone.

Example 2: Vaccination on Mice with the Candidate Antigenic PeptideInduces Improved T Cell Responses in a ELISPOT-IFNγ Assay

A. Materials and Methods

A.1 Mouse Model

The features of the model used are outlined in Table 2:

TABLE 2 Model features. Mouse Model C57BL/6J B2m^(tm1Unc)IAb^(−/−)Tg(HLA-DRA HLA-DRB1*0301)^(#Gjh)Tg(HLA-A/H2-D/B2M)^(1Bpe) Acronym β/A2/DR3 Description Immunocompetent,no mouse class I and class II MHC Housing SOPF conditions (ABSL3) Numberof mice 24 adults (>8 weeks of age)

A.2. Immunization Scheme.

The immunization scheme is shown in FIG. 2. Briefly, 14 (3/A2/DR3 micewere assigned randomly (based on mouse sex and age) to two experimentalgroups, each immunized with a specific vaccination peptide (vacc-pAg)combined to a common helper peptide (h-pAg) (as outlined in Table 3below). The vacc-pAg were compared in couples (group 1 vs. group 2).Thereby, both native and optimized versions of a single peptide werecompared in each wave.

TABLE 3 Experimental group composition. h-pAg: ‘helper’ peptide;vacc-pAg: vaccination peptide. The number of boost injections isindicated into brackets. Peptide Helper Animal Group (vacc-pAg) (h-pAg)Prime Boost number 1 IL13RA2-B HHD-DR3 + + (1X) 6 (100 μg) (150 μg) 2IL13RA2-H HHD-DR3 + + (1X) 6 (100 μg) (150 μg)

The peptides were provided as follows:

-   -   couples of vacc-pAg: IL13RA2-H and IL13RA2-B; all produced and        provided at a 4 mg/ml (4 mM) concentration;    -   h-pAg: HHD-DR3; provided lyophilized (50.6 mg; Eurogentec        batch 1611166) and re-suspended in pure distilled water at a 10        mg/mL concentration; The animals were immunized on day 0 (d0)        with a prime injection, and on d14 with a boost injection. Each        mouse was injected s.c. at tail base with 100 μL of an oil-based        emulsion that contained:    -   100 μg of vacc-pAg (25 μL of 4 mg/mL stock per mouse);    -   150 μg of h-pAg (15 μL of 10 mg/mL stock per mouse);    -   10 μL of PBS to reach a total volume of 50 μL (per mouse);    -   Incomplete Freund's Adjuvant (IFA) added at 1:1 (v:v) ratio (50        μL per mouse).

A separate emulsion was prepared for each vacc-pAg, as follows: IFAreagent was added to the vacc-pAg/h-pAg/PBS mixture in a 15 mL tube andmixed on vortex for repeated cycles of 1 min until forming a thickemulsion.

A.3. Mouse Analysis

Seven days after the boost injection (i.e. on d21), the animals wereeuthanized and the spleen was harvested. Splenocytes were prepared bymechanical disruption of the organ followed by 70 μm-filtering andFicoll density gradient purification.

The splenocytes were immediately used in an ELISPOT-IFNγ assay (Table4). Experimental conditions were repeated in quadruplets, using 2*10⁵total splenocytes per well, and were cultured in presence of vacc-pAg(10 μM), Concanavalin A (ConA, 2.5 μg/mL) or medium-only to assess fortheir capacity to secrete IFNγ. The commercial ELISPOT-IFNγ kit(Diaclone Kit Mujrine IFNγ ELISpot) was used following themanufacturer's instructions, and the assay was performed after about 16h of incubation.

TABLE 4 Setup of the ELISPOT-IFNγ assay. Group Stimulus Wells AnimalTotal 1 IL13RA2-B (10 μM) 4 6 24 IL13RA2-H (10 μM) 4 6 24 ConA (2.5μg/ml) 4 6 24 Medium 4 6 24 2 IL13RA2-B (10 μM) 4 6 24 IL13RA2-H (10 μM)4 6 24 ConA (2.5 μg/ml) 4 6 24 Medium 4 6 24

Spots were counted on a Grand ImmunoSpot® S6 Ultimate UV Image Analyzerinterfaced to the ImmunoSpot 5.4 software (CTL-Europe). Data plottingand statistical analysis were performed with the Prism-5 software(GraphPad Software Inc.).

The cell suspensions were also analyzed by flow cytometry, for T cellcounts normalization. The monoclonal antibody cocktail (data not shown)was applied on the purified leucocytes in presence of Fc-block reagentstargeting murine (1:10 diluted ‘anti-mCD16/CD32 CF11 clone’—internalsource) Fc receptors. Incubations were performed in 96-well plates, inthe dark and at 4° C. for 15-20 minutes. The cells were washed bycentrifugation after staining to remove the excess of monoclonalantibody cocktail, and were re-suspended in PBS for data acquisition.

All data acquisitions were performed with an LSR-II Fortessa flowcytometer interfaced with the FACS-Diva software (BD Bioscience). Theanalysis of the data was performed using the FlowJo-9 software (TreeStarInc.) using a gating strategy (not shown).

TABLE 5 FACS panel EXP-1. Target Label Clone Provider Dilution mCD3εγFITC 145-2C11 Biolegend 1/100 mCD4 PE RM4-5 Biolegend 1/100 mCD8α APC53-6,7 Biolegend 1/100

B. Results

A total of 14 β/A2/DR3 mice were used for this experiment (see Table 6).At time of sacrifice, the spleen T cell population was analysed by flowcytometry, showing that the large majority belonged to the CD4+ T cellsubset.

TABLE 6 Individual mouse features (groups 1 & 2). T Mouse Age^(a) Groupcells^(b) T4^(c) T8^(c) ID Sex (wks) (pAg) (%) (%) (%) Note^(d) 826 M 141 (IL13RA2-B) 18.6 72.0 13.7 P1/2 827 M 14 1 (IL13RA2-B) 21.1 82.5 8.7P1/2 828 M 14 1 (IL13RA2-B) 20.9 78.4 8.6 P1/2 829 F 15 1 (IL13RA2-B)23.8 67.0 17.5 P1/2 830 F 15 1 (IL13RA2-B) 29.2 73.3 12.5 P1/2 831 F 151 (IL13RA2-B) N.A. N.A. N.A. ID tag lost (excluded) 17 M 9 1 (IL13RA2-B) 8.3 83.7 10.4 P5 832 F 15 2 (IL13RA2-H) 28.3 83.4 5.7 P1/2 833 F 15 2(IL13RA2-H) N.A. N.A. N.A. ID tag lost (excluded) 834 F 15 2 (IL13RA2-H)27.5 79.7 7.2 P1/2 835 M 13 2 (IL13RA2-H) 33.8 84.2 8.5 P1/2 836 M 13 2(IL13RA2-H) 31.4 84.7 6.3 P1/2 837 M 15 2 (IL13RA2-H) 30.8 83.4 5.4 P1/218 M 9 2 (IL13RA2-H) 11.2 85.9 9.2 P5 Each mouse is identified by aunique ear tag ID number. ^(a)age at onset of the vaccination protocol(in weeks); ^(b)percentage of T cells in total leukocytes;^(c)percentage of CD4+ or CD8+ T cells in total T cells; ^(d)plate (P)number.

After plating and incubation with the appropriate stimuli, theIFNγ-producing cells were revealed and counted. The data were thennormalized as a number of specific spots (the average counts obtained inthe ‘medium only’ condition being subtracted) per 10⁶ total T cells.

The individual average values (obtained from the quadruplicates) werenext used to plot the group average values (see FIG. 3A). As thefunctional capacity of T cells might vary from individual to individual,the data were also expressed as the percentage of the ConA response perindividual (see FIG. 3B).

Overall, vaccination with the IL13RA2-B pAg (candidate) peptide inducedimproved T cell responses in the ELISPOT-IFNγ assay, as compared toIL13RA2-H pA (reference human)-vaccinated animals (group 2). For group 1(IL13RA2-B), ex vivo re-stimulation with the IL13RA2-B pAg promotedhigher response than with the IL13RA2-H pAg. It was not the case forgroup 2 (IL13RA2-H). The percentage of ConA-induced response(mean+/−SEM) for each condition was as follows:

-   -   Group 1 (IL13RA2-B)/IL13RA2-B pAg: 56.3%+/−18.1    -   Group 1 (IL13RA2-B)/IL13RA2-H pAg: 32.3%+/−11.8    -   Group 2 (IL13RA2-H)/IL13RA2-B pAg: 2.0%+/−0.8    -   Group 2 (IL13RA2-H)/IL13RA2-H pAg: 1.1%+/−0.8

Accordingly, those results provide experimental evidence thattumor-antigen immunotherapy targeting IL13RA2 is able to improve T cellresponse in vivo and that the IL13RA2-B candidate peptide (SEQ ID NO:71)is particularly efficient for that purpose.

Example 3: Identification of Further Candidate Antigenic Peptides HavingSuperior Affinity to the HLA-A*0201 Allele

This Example provides evidence that the antigenic peptides of sequenceSEQ ID NO:47 («RLLEETDLV» also referred herein as ERBB2-1B); SEQ IDNO:51 («VMLGVVFGV» also referred herein as ERBB2-3B1); SEQ ID NO:52(«VLLGVVFGV» also referred herein as ERBB2-3B2); SEQ ID NO:55(«VMLGVVFGI» also referred herein as ERBB2-3B3); and SEQ ID NO:56(«ILLGVVFGI» also referred herein as ERBB2-3B4) have higher affinity tothe HLA-A*0201 allele than the corresponding reference human peptidesderived from ERBB2 («RLLQETELV», SEQ ID NO:120, also referred herein asERBB2-1H; («VVLGVVFGI», SEQ ID NO:122, also referred herein asERBB2-3H).

A. Materials and Methods

A1. Measuring the Affinity of the Peptide to T2 Cell Line.

The experimental protocol is similar to the one that was validated forpeptides presented by the HLA-A*0201 (Tourdot et al., A general strategyto enhance immunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification of cryptic tumor epitopes. Eur JImmunol. 2000 December; 30(12):3411-21). Affinity measurement of thepeptides is achieved with the human tumoral cell T2 which expresses theHLA-A*0201 molecule, but which is TAP1/2 negative and incapable ofpresenting endogenous peptides.

T2 cells (2·10⁵ cells per well) are incubated with decreasingconcentrations of peptides from 100 μM to 0.1 μM in a AIMV mediumsupplemented with 100 ng/μl of human β2m at 37° C. for 16 hours. Cellsare then washed two times and marked with the anti-HLA-A2 antibodycoupled to PE (clone BB7.2, BD Pharmagen).

The analysis is achieved by FACS (Guava Easy Cyte).

For each peptide concentration, the geometric mean of the labellingassociated with the peptide of interest is subtracted from backgroundnoise and reported as a percentage of the geometric mean of theHLA-A*0202 labelling obtained for the reference peptide HIV pol 589-597at a concentration of 100 μM. The relative affinity is then determinedas follows:relative affinity=concentration of each peptide inducing 20% ofexpression of HLA-A*0201/concentration of the reference peptide inducing20% of expression of HLA-A*0201.

A2. Solubilisation of Peptides

Each peptide is solubilized by taking into account the amino acidcomposition. For peptides which do not include any Cystein, Methionin,or Tryptophane, the addition of DMSO is possible to up to 10% of thetotal volume. Other peptides are re-suspended in water or PBS pH7.4.

B. Results

Results are shown in Table 7:

Conc. inducing SEQ 20% of HLA-A2 Relative Peptide ID NO. 100 μM 10 μM 1μM 0.1 μM expression [μM] affinity ERBB2-1B 47 296.97 26.39 2.86 −1.189.5 0.26 ERBB2-1H 120 108.74 15.63 −5.21 −5.88 16.3 0.45 ERBB2-3B1 51122.18 26.72 −12.94 −15.97 9 0.25 ERBB2-3B2 52 335.97 56.97 1.51 −14.626.9 0.19 ERBB2-3B3 55 178.66 16.64 −10.59 −16.3 12.5 0.35 ERBB2-3B4 56265.38 138.32 26.05 −11.6 0.9 0.03 ERBB2-3H 122 196.47 11.93 −24.03−12.61 16.3 0.45 HIV pol 589-597 100 −3.03 −5.38 −9.24 36

As shown in Table 7 (see, in particular, “relative affinity” calculatedas described above), antigenic peptide ERBB2-1B shows higher affinity(lower value) than the corresponding human peptide ERBB2-1H. Moreover,antigenic peptides ERBB2-3B1, ERBB2-3B2, ERBB2-3B3 and ERBB2-3B4 showhigher affinity (lower value) than the corresponding human peptideERBB2-1B. Moreover, lower concentrations of the antigenic peptidesERBB2-1B, ERBB2-3B1, ERBB2-3B2, ERBB2-3B3 and ERBB2-3B4 (as compared tothe human reference peptides) are required to induce 20% of expressionof the HLA-A*0201.

Similar results were obtained from a second distinct T2 cell clone.

Example 4: Identification of Further Candidate Antigenic Peptides HavingSuperior Affinity to the HLA-A*0201 Allele

This Example provides evidence that the antigenic peptides of sequenceSEQ ID NO:77 («KLVEWLAML» also referred herein as MAGE CIB); SEQ IDNO:93 («SLPPDVQQV» also referred herein as MMP2-B); SEQ ID NO:28(«ITSDVPFSV» also referred herein as PMEL-B); SEQ ID NO:101 («MLAVFLPLV»also referred herein as STEAP-B1); and SEQ ID NO:102 («YLAVFLPIV» alsoreferred herein as STEAP-B2) have higher affinity to the HLA-A*0201allele than the corresponding reference human peptides derived from MAGEC1 («KVVEFLAML», SEQ ID NO:128, also referred herein as MAGE C1H), MMP2(«GLPPDVQRV», SEQ ID NO:136, also referred herein as MMP2-H), PMEL(«ITDQVPFSV», SEQ ID NO:115, also referred herein as PMEL-H), and STEAP(«MIAVFLPIV», SEQ ID NO: 141, also referred herein as STEAP-H).

A. Materials and Methods

A1. Measuring the Affinity of the Peptide to T2 Cell Line.

The experimental protocol is similar to the one that was validated forpeptides presented by the HLA-A*0201 (Tourdot et al., A general strategyto enhance immunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification of cryptic tumor epitopes. Eur JImmunol. 2000 December; 30(12):3411-21). Affinity measurement of thepeptides is achieved with the human tumoral cell T2 which expresses theHLA-A*0201 molecule, but which is TAP1/2 negative and incapable ofpresenting endogenous peptides.

T2 cells (2·10⁵ cells per well) are incubated with decreasingconcentrations of peptides from 100 μM to 0.1 μM in a AIMV mediumsupplemented with 100 ng/μl of human β2m at 37° C. for 16 hours. Cellsare then washed two times and marked with the anti-HLA-A2 antibodycoupled to PE (clone BB7.2, BD Pharmagen).

The analysis is achieved by FACS (Guava Easy Cyte).

For each peptide concentration, the geometric mean of the labellingassociated with the peptide of interest is subtracted from backgroundnoise and reported as a percentage of the geometric mean of theHLA-A*0202 labelling obtained for the reference peptide HIV pol 589-597at a concentration of 100 μM. The relative affinity is then determinedas follows:relative affinity=concentration of each peptide inducing 20% ofexpression of HLA-A*0201/concentration of the reference peptide inducing20% of expression of HLA-A*020L.

A2. Solubilisation of Peptides

Each peptide is solubilized by taking into account the amino acidcomposition. For peptides which do not include any Cystein, Methionin,or Tryptophane, the addition of DMSO is possible to up to 10% of thetotal volume. Other peptides are re-suspended in water or PBS pH7.4.

B. Results

Results are shown in Table 8:

Conc. inducing SEQ 20% of HLA-A2 Relative Peptide ID NO. 100 μM 10 μM 1μM 0.1 μM expression [μM] affinity MAGE C1B 77 108.8 21.4 3.97 2.4530.91 0.31 MAGE C1H 128 32.27 7.84 7.12 5.77 60.07 1.94 MMP2-B 93 131.0895.96 24.64 4.69 0.88 0.03 MMP2-H 136 154.17 66.31 17.81 5.41 1.76 0.06PMEL-B 28 74.85 7.93 3.62 4.69 18.24 0.59 PMEL-H 115 112.58 9.09 5.321.01 23.94 0.77 STEAP-B1 101 131.62 45.12 8.92 6.67 5 0.16 STEAP-B2 10297.93 27.69 4.87 −0.34 8.22 0.27 STEAP-H 141 101.98 14.93 −4.47 0.1133.45 1.08 HIV pol 589-597 100 3.8 −2.54 2.58 30.91

As shown in Table 8 (see, in particular, “relative affinity” calculatedas described above), antigenic peptide MAGE C1B shows higher affinity(lower value) than the corresponding human peptide MAGE C1H. Moreover,antigenic peptide MMP2-B shows higher affinity (lower value) than thecorresponding human peptide MMP2-H. Moreover, antigenic peptide PMEL-Bshows higher affinity (lower value) than the corresponding human peptidePMEL-H. Moreover, antigenic peptides STEAP-B1 and STEAP-B2 show higheraffinity (lower value) than the corresponding human peptide STEAP-H.Moreover, lower concentrations of the antigenic peptides MAGE C1B,MMP2-B, PMEL-B, STEAP-B1 and STEAP-B2 (as compared to their humanreference peptides) are required to induce 20% of expression of theHLA-A*0201.

Similar results were obtained from a second distinct T2 cell clone.

Example 5: Identification of Further Candidate Antigenic Peptides HavingSuperior Affinity to the HLA-A*0201 Allele

This Example provides evidence that the antigenic peptide of sequenceSEQ ID NO:26 («TMNGKSSPV» also referred herein as ENAH-B) has highaffinity to the HLA-A*0201 allele, whereas the corresponding referencehuman peptide derived from ENAH («TMNGSKSPV», SEQ ID NO: 113, alsoreferred herein as ENAH-H) has low affinity.

A. Materials and Methods

A1. Measuring the Affinity of the Peptide to T2 Cell Line.

The experimental protocol is similar to the one that was validated forpeptides presented by the HLA-A*0201 (Tourdot et al., A general strategyto enhance immunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification of cryptic tumor epitopes. Eur JImmunol. 2000 December; 30(12):3411-21). Affinity measurement of thepeptides is achieved with the human tumoral cell T2 which expresses theHLA-A*0201 molecule, but which is TAP1/2 negative and incapable ofpresenting endogenous peptides.

T2 cells (2·10⁵ cells per well) are incubated with decreasingconcentrations of peptides from 100 μM to 0.1 μM in a AIMV mediumsupplemented with 100 ng/μl of human β2m at 37° C. for 16 hours. Cellsare then washed two times and marked with the anti-HLA-A2 antibodycoupled to PE (clone BB7.2, BD Pharmagen).

The analysis is achieved by FACS (Guava Easy Cyte).

For each peptide concentration, the geometric mean of the labellingassociated with the peptide of interest is subtracted from backgroundnoise and reported as a percentage of the geometric mean of theHLA-A*0202 labelling obtained for the reference peptide HIV pol 589-597at a concentration of 100 μM. The relative affinity is then determinedas follows:relative affinity=concentration of each peptide inducing 20% ofexpression of HLA-A*0201/concentration of the reference peptide inducing20% of expression of HLA-A*0201.

A2. Solubilisation of Peptides

Each peptide is solubilized by taking into account the amino acidcomposition. For peptides which do not include any Cystein, Methionin,or Tryptophane, the addition of DMSO is possible to up to 10% of thetotal volume. Other peptides are re-suspended in water or PBS pH7.4.

B. Results

Results are shown in Table 9:

Conc. inducing SEQ 20% of HLA-A2 Relative Peptide ID NO. 100 μM 10 μM 1μM 0.1 μM expression [μM] affinity ENAH-1B 26 100.24 2.93 14.18 12.7133.45 1.26 ENAH-1H 113 18.58 19.07 −2.93 8.31 ND ND HIV pol 589-597 1008.8 4.65 5.62 26.48

As shown in Table 9 (see, in particular, “relative affinity” calculatedas described above), antigenic peptide ENAH-B shows higher affinity(lower value) than the corresponding human peptide ENAH-H. Inparticular, it appears that the human peptide ENAH-H does not bind tothe HLA-A*0201 (ND . . . not determined).

Moreover, lower concentrations of the antigenic peptide ENAH-B (ascompared to the human reference peptide ENAH-H) were required to induce20% of expression of the HLA-A*0201.

Similar results were obtained from a second distinct T2 cell clone.

The invention claimed is:
 1. An immunogenic compound, comprising anantigenic peptide of formula (I):PepNt-CORE-PepCt  (I), wherein: “PepNt” consists of a polypeptide havingan amino acid length varying from 0 to 500 amino acid residues andlocated at the N-terminal end of the polypeptide of formula (I); COREconsists of a polypeptide comprising, or alternatively consisting of, anamino acid sequence selected from the group consisting of SEQ ID NO: 100and SEQ ID NO: 101; and “PepCt” consists of a polypeptide having anamino acid length varying from 0 to 500 amino acid residues and locatedat the C-terminal end of the polypeptide of formula (I); wherein saidimmunogenic compound comprises a carrier protein.
 2. A nanoparticleloaded with at least one immunogenic compound comprising an antigenicpeptide, wherein the antigenic peptide comprises or consists of an aminoacid sequence as set forth in SEQ ID NO:100 or SEQ ID NO:101, or atleast one antigenic peptide comprising or consisting of an amino acidsequence as set forth in SEQ ID NO:100 or SEQ ID NO:101; and,optionally, with an adjuvant.
 3. A vector comprising a polynucleotide,said polynucleotide encoding: an immunogenic compound comprising anantigenic peptide, wherein the antigenic peptide comprises or consistsof an amino acid sequence as set forth in SEQ ID NO: 100 or SEQ IDNO:101, wherein the immunogenic compound is a peptide or a protein; oran antigenic peptide comprising or consisting of an amino acid sequenceas set forth in SEQ ID NO:100 or SEQ ID NO:101.
 4. A host cellcomprising the vector comprising a polynucleotide according to claim 3.5. The host cell according to claim 4, wherein the host cell is abacterial cell.
 6. An immunogenic composition comprising: (i) animmunogenic compound comprising a first antigenic peptide, wherein thefirst antigenic peptide comprises or consists of an amino acid sequenceas set forth in SEQ ID NO:100 or SEQ ID NO:101, (ii) the first antigenicpeptide comprising or consisting of an amino acid sequence as set forthin SEQ ID NO: 100 or SEQ ID NO:101, (iii) a nanoparticle loaded with atleast one immunogenic compound of (i) or the first antigenic peptide of(ii), (iv) a cell loaded with the immunogenic compound of (i) or thefirst antigenic peptide of (ii), (v) a nucleic acid comprising apolynucleotide encoding (a) the immunogenic compound of (i), wherein theimmunogenic compound is a peptide or a protein, or (b) the firstantigenic peptide of (ii), or (vi) a host cell comprising the nucleicacid of (v); and one or more immunostimulatory agents, wherein saidimmunostimulatory agents are each independently selected from the groupconsisting of: immunoadjuvants and antigen-presenting cells; and one ormore pharmaceutically acceptable excipients.
 7. The immunogeniccomposition according to claim 6, wherein the composition comprises (i)two distinct immunogenic compounds each comprising an antigenic peptide,wherein the first antigenic peptide comprises or consists of an aminoacid sequence as set forth in SEQ ID NO:100 or SEQ ID NO:101 and asecond antigenic peptide comprises or consists of an amino acid sequenceas set forth in any one of SEQ ID NO:1-70 and 72-106; (ii) two distinctantigenic peptides, wherein the first antigenic peptide comprises orconsists of an amino acid sequence as set forth in SEQ ID NO:100 or SEQID NO:101 and a second antigenic peptide comprises or consists of anamino acid sequence as set forth in any one of SEQ ID NO:1-70 and72-106; (iii) two distinct nanoparticles loaded with the immunogeniccompounds of (i) or the antigenic peptides of (ii); or (iv) two distinctnucleic acids comprising a polynucleotide encoding (a) the immunogeniccompounds of (i), wherein the immunogenic compounds are peptides orproteins, or (b) the antigenic peptides of (ii).
 8. A method forreducing the occurrence of and/or treating a cancer; or initiating,enhancing or prolonging an anti-tumor-response in a subject in needthereof comprising administering to the subject: (i) an immunogeniccompound comprising an antigenic peptide, wherein the antigenic peptidecomprises or consists of an amino acid sequence as set forth in SEQ IDNO:100 or SEQ ID NO:101, (ii) an antigenic peptide comprising orconsisting of an amino acid sequence as set forth in SEQ ID NO:100 orSEQ ID NO:101, (iii) a nanoparticle loaded with at least one immunogeniccompound of (i) or at least one antigenic peptide of (ii), (iv) a cellloaded with the immunogenic compound of (i) or the antigenic peptide of(ii), (v) a nucleic acid comprising a polynucleotide encoding (a) theimmunogenic compound of (i), wherein the immunogenic compound is apeptide or a protein, or (b) the antigenic peptide of (ii), (vi) a hostcell comprising the nucleic acid of (v), (vii) an immunogeniccomposition comprising the immunogenic compound of (i), the antigenicpeptide of (ii), the nanoparticle of (iii), the cell of (iv), thenucleic acid of (v), or the host cell of (vi), or (viii) a combinationof at least two distinct antigenic peptides, the combination comprisingthe antigenic peptide of (ii) and at least one antigenic peptide ofinterest; and wherein the subject has an HLA-A2 allele and a tumorexpressing a six transmembrane epithelial antigen of the prostate(STEAP) gene.
 9. The method according to claim 8, wherein the cancer isselected from the group consisting of prostate cancer, melanoma,colorectal cancer and clear cell renal cell carcinoma.
 10. The vectorcomprising a polynucleotide according to claim 3, wherein thepolynucleotide is a DNA sequence with or without expression elements,regulatory elements, and/or promoters; or a combination thereof.
 11. Thehost cell according to claim 5, wherein the bacterial cell is a gutbacterial cell.